Polymer conjugated protein micelles

ABSTRACT

The invention encompasses micelle assemblies, compositions having micelle assemblies, and methods for preparing micelle assemblies and compositions thereof. The invention also encompasses a prolamine protein conjugated to a polymer, such as a polyethylene glycol (PEG) chain, which conjugates can be used to prepare micelle assemblies. The invention further encompasses methods of encapsulating molecules using the conjugates of the invention. The micelle assemblies can be used for a variety of applications, such as treating cancer, targeting tumors, reducing the toxicity of a drug in vivo, increasing the efficacy of an encapsulated agent in vivo, protecting an encapsulated agent against degradation, and enhancing the water solubility of a drug or other agent.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/446,935, filed Feb. 25, 2011, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to drug delivery technologies,and more specifically to a nanomicelle drug delivery system, includingmethods for preparing such a system using a hydrophobic water insolubleprotein and a water soluble polymer, which micelles may includepolyethylene glycol (PEG) or other hydrophilic moieties that may becovalently attached to hydrophobic water insoluble proteins such asprolamines to form an amphiphilic conjugate for preparing thenanomicelle drug delivery system.

BACKGROUND INFORMATION

Approximately 40% of pharmaceutical compounds have poor aqueoussolubility, which is a major limiting factor for a new drug tosuccessfully pass through clinical trials (Lipinski (2002), Am Pharm Rev5:82-85). Numerous approaches have been used to solubilize hydrophobicdrugs for improving their delivery to patients. Several examples of suchapproaches include milling, complexing with cyclodextrins, formingsalts, and using surfactants or polymeric micelles. Each of theseapproaches has certain advantages and disadvantages so improvedapproaches to solubilizing drugs are eagerly sought.

Polymeric micelles are self-assembled amphiphilic block or graftcopolymers. Polymeric micelles have attracted attention as promisingcolloidal drug delivery systems (Torchilin, J Controlled Release 2001,73, 137; Allen et al., Colloids and Surfaces B: Biointerfaces 1999, 16,3; and Otsuka et al., Current Opinion in Colloid & Interface Science2001, 6, 3). In these colloidal systems, the hydrophobic block typicallyforms the core, essentially forming a “microcontainer” for a lipophiliccargo molecules (Kataoka et al., Adv. Drug Delivery Rev. 2001, 47, 113).The hydrophilic portion of the micelle forms the outer shell,stabilizing the interface between the core and the external aqueousenvironment.

Compared to surfactant-based micellar systems, polymer-based micellescan display apparent advantages such as lower critical micelleconcentration (CMC) and reduced toxicity. Despite these advantages, theuse of known micellar systems is somewhat limited due to unsuitablebiodegradability, biocompatibility, encapsulation efficiency, stability,clinical side effects of the formulations, and the difficulty and costassociated with preparation of known micellar formulations. Accordingly,there is a need for additional micellar systems that possess some of theknown advantages associated with micellar drug delivery systems, butthat have increased biocompatibility and are easier and less expensiveto prepare.

SUMMARY OF THE INVENTION

The invention provides a novel nanomicelle platform technology fordelivery of water insoluble compounds. Also provided are methods forpreparing micelles using a hydrophobic water insoluble protein and awater soluble polymer, and methods of using micelle compositions, forexample, for in vivo drug delivery. Polyethylene glycol (PEG) or otherhydrophilic moieties can be covalently attached to hydrophobic waterinsoluble proteins such as zein, to form a highly useful amphiphilicconjugate for preparing the nanomicelle drug delivery systems.

Accordingly, the invention provides a micelle comprising biocompatibleand biodegradable copolymers, wherein the copolymers include ahydrophobic block and a hydrophilic block; the hydrophobic blockincludes a hydrophobic prolamine protein covalently conjugated to thehydrophilic block and the hydrophilic block includes a hydrophilicpolyethylene glycol moiety having a molecular weight of at least about 3kDa; prolamine protein chains of the amphiphilic copolymers orienttoward the interior of the micelle, and polyethylene glycol moiety ofthe amphiphilic copolymers orient toward the exterior of the micelle;and the diameter of the micelle is about 10 nm to about 300 nm.

The biocompatible and biodegradable copolymers may in include graftcopolymers and/or block copolymers. The critical micelle concentrationof the micelle in water can be about 0.015 g/L to about 0.035 g/L, about0.02 g/L to about 0.03 g/L, or about 0.25 g/L, for example, at about 27°C. The hydrophobic drug can have a Log P of about 1, about 2, about 3,about 4, about 5, about 6, about 7, or about 1 to about 7, or a rangefrom any integer from 1 to 7.

The hydrophobic prolamine protein can be zein, gliadin, hordein,kafirin, or a combination thereof. The hydrophilic polyethylene glycolmoiety can have a molecular weight of about 4 kDa to about 220 kDa. Thehydrophilic polyethylene glycol moiety can have a molecular weight ofabout 4 kDa to about 20 kDa.

The micelles can further include a plurality of cargo molecules in thecore of the micelle. The cargo molecules can include, for example, oneor more drugs, proteins, nucleic acids, hormones, receptors, diagnosticagents, imaging agents, or a combination thereof. The drug can be anantioxidant, an anti-inflammatory drug or an anticancer drug. In oneembodiment, the drug is curcumin or doxorubicin. In another embodiment,the imaging agent is Nile red.

In another embodiment, the drug is a retinoid. Examples of suitableretinoids include retinol, 13-trans-retinoic acid (tretinoin),13-cis-retinoic acid (isotretinoin), 9-cis-retinoic acid (alitretinoin),retinaldehyde, etretnate, acitretin, α-carotene, β-carotene, γ-carotene,β-cryptozanthin, lutein, zeaxanthin, or a combination thereof.

The invention also provides a pharmaceutical or cosmetic compositioncomprising a plurality of micelles described herein and apharmaceutically or cosmetically acceptable diluent, excipient, orcarrier. The pharmaceutical or cosmetic composition may be, for example,in the form of a dispersion, tablet, capsule, injectable formulation,aerosol formulation, gel, ointment, cream, lotion, or shampoo.

The invention further provides a method of preparing a micelle. Themethod may include adding a buffer to an aqueous suspension toprecipitate PEGylated prolamine from a hydroalcoholic solvent to form anaqueous dispersion of PEGylated prolamine, and removing the alcohol andunreacted PEG and glycine in the dispersion by dialysis againstdeionized water.

The invention additionally provides a method of preparing a micellewherein the method includes removing alcohol from an aqueous suspensionof PEGylated prolamine to form a dry film of PEGylated prolamine; andresuspending the PEG-zein film in water or a buffer followed by dialysisagainst deionized water, for example, to remove unencapsulatedhydrophobic molecules, to provide a plurality of the micelles, forexample, in a water and buffer composition, such as a dispersion. Inthis method, after PEGylation of zein, the ethanol can be removed byevaporation, for example, in a rotary evaporator, to form a dry film.The dry film can then be reconstituted in water and dialyzed againstdeionized water, for example, to remove unencapsulated hydrophobiccompounds, to form micelles in the aqueous phase.

Accordingly, the invention also provides a method of preparing themicelles described herein by dissolving a polyethylene glycol compoundand a prolamine protein in a hydroalcoholic solvent to form a firstmixture; wherein one terminus of the polyethylene glycol compound ismonoalkylated and a second terminus comprises a reactive group, and thepolyethylene glycol compound has a molecular weight of at least about 3kDa; heating the first mixture to form PEGylated prolamine in an aqueoussuspension, and optionally quenching excess reactive groups of thepolyethylene glycol compound in the aqueous suspension and removingalcohol and unreacted PEG and glycine in the dispersion by dialysisagainst deionized water followed by lyophilization.

The remainder of the method can follow one of two paths. In oneembodiment, the method includes (a) adding PEG-zein in a hydroalcoholicsolvent and removing alcohol by dialysis against deionized water to forma plurality of micelles in a buffer and water composition. In anotherembodiment, the method includes (b) removing the alcohol of the aqueoussuspension to form a dry film of PEGylated prolamine; and resuspendingthe PEGylated prolamine of the dry film in water or a buffer followed bydialysis, for example, to remove unencapsulated hydrophobic compounds,to form micelles in the aqueous phase.

Thus, once prepared, the PEG-prolamine can be dissolved inhydroalcoholic solution at a specific concentration. When following thefilm method of preparation, the alcohol is removed to form a PEG-zeinfilm. The film is then reconstituted with deionized water to formPEG-zein micelles. This composition can then be dialyzed against waterto remove unencapsulated compounds. Formation of the aqueous dispersioncan then be followed by lyophilization to obtain a PEG-zein micellepowder.

When following the dialysis method of preparation, the alcohol isremoved by dialyzing against deionized water to form micelles. Theaqueous dispersion may then be lyophilized to provide a PEG-zein micellepowder. Accordingly, the methods may include lyophilizing a plurality ofthe micelles in a composition such as a dispersion to provide aplurality of isolated micelles in the form of a powder.

In any of the methods of preparation, useful cargo molecules, forexample, a therapeutic agent or an imaging agent can be dissolved in asolvent system and can be added to the first mixture, resulting in theformation of a cargo loaded micelles, such as drug loaded micelles. Theencapsulation efficiency of the micelles can be about 60% to about 95%.The method can include dispersing a plurality of the micelles in abuffer solution to provide a therapeutic composition of drug loadedmicelles.

In one embodiment, the invention provides a method to inhibit cellularP-glycoprotein (P-pg) efflux pumps in a cell comprising contacting acell with a plurality of micelles described herein, thereby inhibitingthe cellular P-pg efflux pumps in the cell.

In another embodiment, the invention provides a method to enhance theuptake of a therapeutic agent in a drug-resistance cancer cellcomprising contacting the cell with a plurality of micelles describedherein, thereby enhancing the uptake of the therapeutic agent in thedrug-resistance cancer cell.

In another embodiment, the invention provides a method to enhance thewater solubility of a lipophilic compound comprising encapsulating thelipophilic compound in a micelle as described herein, thereby enhancingthe water solubility of the lipophilic compound.

In another embodiment, the invention provides a method to enhance thechemical stability of a compound comprising encapsulating the compoundin a micelle as described herein, thereby enhancing the chemicalstability of the compound.

In another embodiment, the invention provides a method to providesustained release of a compound from a composition includingencapsulating a compound in a micelle as described herein and contactinga biological medium with the encapsulated compound, where the compoundis released from the micelle over a period of about 1 hour to about 14days.

In another embodiment, the invention provides a method to provide acompound to a subject or a sample in a non-immunogenic and biocompatibleformulation comprising contacting the subject or the sample with amicelle or a composition described herein, thereby providing thenon-immunogenic and biocompatible formulation to the subject or thesample. In one aspect, such formulation may improve systemic circulationof the encapsulated compound.

In another embodiment, the invention provides a method to increase theskin penetration and retention of an active agent or imaging agentcomprising encapsulating the active agent or imaging agent in a micelleas described herein and contacting skin with a composition comprisingthe micelle, thereby increasing the skin penetration of the active agentor imaging agent compared to the skin penetration of the active agent orimaging agent in the absence of the micelle.

In another embodiment, the invention provides a method to enhanced tumoraccumulation of drug comprising encapsulating a drug in a micelle asdescribed herein, and administering to a subject that has a tumor aplurality of the micelles, where the encapsulated drug accumulates atthe tumor to a greater degree than a drug that is administered to asubject in the absence of the micelles.

In another embodiment, the invention provides a method to reduced drugaccumulation in non-tumor bearing tissues in a mammal comprisingencapsulating a drug in a micelle as described herein, and administeringto a subject that has a tumor a plurality of the micelles, where theencapsulated drug accumulates in non-tumor bearing tissues to a lesserdegree than a drug that is administered to a subject in the absence ofthe micelles.

In another embodiment, the invention provides a method to increase theefficacy of a drug comprising administering a plurality of loadedmicelles as described herein to a subject, where the efficacy of thedrug is increased compared to administration of the drug in the absenceof the micelles.

In yet another embodiment, the invention provides a method to reduce thetoxicity of a drug comprising administering a plurality of loadedmicelles as described herein to a subject, where the toxicity of thedrug is reduced compared to administration of the drug in the absence ofthe micelles.

The invention also provides a copolymer of Formula I:

Z-(PEG)_(n)  (I)

where Z is a prolamine protein, “PEG” is a polyethylene glycol moietyhaving a molecular weight of at least about 3 kDa, and n is about 1 toabout 100, or about 5 to about 50. The prolamine protein can be, forexample, white zein, yellow zein, gliadin, hordein, or kafirin. Avariety of PEG moieties with varying molecular weights can be conjugatedto the prolamine For example, molecular weight of the PEG moiety may be1 kDa to about 220 kDa, about 2 kDa to about 20 kDa, about 3 kDa toabout 20 kDa, about 4 kDa to about 20 kDa, about 4 kDa to about 10 kDa,or about 5 kDa.

Formula I can include graft copolymers of a prolamine and PEG; and blockcopolymers of a prolamine and PEG (diblock or multiblock copolymers,such as triblock copolymers). Examples include Formulas II-V:

Z-g-PEG (graft copolymer)  (II)

Z-b-PEG (diblock copolymer)  (III)

Z-b-PEG-b-Z (triblock copolymer)  (IV)

PEG-b-Z-b-PEG (triblock copolymer)  (V)

Copolymers of Formula I are useful intermediates for preparingaggregates that can be used in drug delivery applications, such as forthe delivery of hydrophobic therapeutic agents.

The invention further provides a composition comprising a plurality ofcopolymers or micelles as described herein in a liquid suspension,dispersion or solution, such as an I.V. formulation.

The invention also provides a method for preparing an encapsulate of theinvention comprising combining a plurality of copolymers of Formula Iand a molecule (e.g., a therapeutic agent) in a solvent or solventsystem, and allowing the copolymers of Formula Ito aggregate around themolecule, to provide the encapsulate (i.e. the molecule encapsulated orsurrounded by a plurality of copolymers of Formula I).

The invention also provides a composition comprising a diluent and amicelle formed from a plurality of copolymers of Formula I surrounding amolecule (e.g., a therapeutic agent).

The invention also provides a pharmaceutical composition comprising anencapsulate of the invention (i.e., a therapeutic agent encapsulated orsurrounded by a plurality of copolymers of Formula I, such as in amicelle); and a pharmaceutically acceptable carrier. Alternatively, thetherapeutic agent may be conjugated or complexed to the prolamine in thehydrophobic core and/or to the hydrophilic polymeric shell. The micellecan also be used to encapsulate multiple therapeutic agents, and/ormultiple therapeutic agents can be complexed/conjugated to the coreand/or shell. In addition to or in place of therapeutic agents, themicelles can be used to carry diagnostic and/or imaging agents, and thelike.

The invention also provides a method for delivering a therapeutic agentto an animal in need of treatment with the agent comprisingadministering an encapsulate of the invention to the animal, where theencapsulate includes the therapeutic agent inside a micellar assembly ofcopolymers of Formula I.

The invention further provides for the use of the micellar compositionsdescribed herein for use in medical therapy. The medical therapy can betreating cancer, for example, breast cancer, lung cancer, pancreaticcancer, prostate cancer, or colon cancer. The invention also providesfor the use of a micellar composition as described herein for themanufacture of a medicament to treat such cancers. The medicament caninclude a pharmaceutically acceptable diluent, excipient, or carrier.

The invention further provides for the treatment of skin and folliculardisorders, such as acne, and may be used in the treatment of hair loss,seborrhetic eczema, follicullitis, cutaneous malignancies, psoriasis,keratinization disorders, skin discoloration, wounds, and photoaging.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention, however, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 schematically illustrates the formation of drug loaded PEG-zeinnanomicelles, according to an embodiment.

FIG. 2 illustrates a flow chart depicting general steps for preparationof amphiphilic PEG-Zein, according to an embodiment.

FIG. 3 illustrates a flow chart depicting specific steps for preparationof amphiphilic PEG-Zein, according to one embodiment. The specificamounts recited in this and other figures are for illustration of aparticular embodiment, and many variations can be applied to theprocedures described herein, as would be readily recognized by oneskilled in the art.

FIG. 4 illustrates characterization of PEG-zein conjugate by (a) FTIRand (b) size exclusion chromatography. FTIR spectrum of zein, PEG-ester,and PEG-zein (5 mg each) were recorded on ZnSe crystal at 2 cm⁻¹resolution in NICOLET 380 ATR-FTIR spectrophotometer (THERMO ELECTRONCorporation, Madison, Wis.). Each spectrum was an average of 100 scans.The peak position of functional groups was analyzed using OMNICsoftware. SEC was carried out using a PHENOMENEX BISEP SEC-S 2000 4.6mm×3000 column (PHENOMENEX®, Torrance, Calif.) in a HPLC (BECKMANCOULTER, Brea, Calif.) system. The samples were separated using 70%(v/v) ethanol as mobile phase using a flow rate of 0.5 mL/min. Thecolumn eluate was monitored at 280 nm.

FIG. 5 depicts the ¹H NMR spectrum of PEG-Zein in (a) DMSO and (b) D₂O.In FIG. 5( a), ethylene groups of PEG are observed at 3.56 ppm and theprotein/amide peak is observed at 3.36 ppm. In FIG. 5( b), ethylenebonds of PEG are observed at 3.56 ppm, while the protein/amide peak at3.36 ppm is absent because the hydrophobic zein core is not soluble inD₂O.

FIG. 6 illustrates the stability of PEG-Zein micelles upon dilution; 2mg/mL stock dispersion of PEG-zein micelles in 10 mM citrate buffer pH7.4 was diluted 20, 100, 200, 500, and 1000 times, and the size of themicelles was determined in a particle size analyzer (NICOMP 380 ZLS ZetaPotential Analyzer, Particle Sizing Systems, Santa Barbara, Calif.). Thedata show that the micelles were stable upon dilution because the sizeof the micelles did not change significantly. Each data point is a meanof three experiments±SD.

FIG. 7 illustrates the plot of the ratio of absorbance of pyrene (0.6μM) at the excitation wavelengths of 339 nm and 334 nm (emissionwavelength is 390 nm) against logarithmic concentration (g/L) ofPEGylated zein. As the concentration of PEGylated zein is increased, theintensity of absorbance of pyrene at the critical micellar concentration(CMC) shifts significantly. The CMC for PEGylated zein is 0.025 (g/L) at27° C.

FIG. 8 illustrates the immune response after in vivo administration ofPEG-zein micelles in mice. Anti-zein antibodies (optical density iny-axis) in serum was measured after the third week of the first dose andthe 5th week after the booster dose. Saline or PEG-Zein micelles (100μg/50 μL) was administered subcutaneously in mice. The results arerepresented as mean±standard error of mean (n=4). The PEG-zein micellesdid not produce any anti-zein antibodies and the values were similar tothe saline control.

FIG. 9 illustrates steps of preparing curcumin-loaded PEGylated zeinmicelles using a film method, according to an embodiment.

FIG. 10 illustrates steps of preparing curcumin-loaded PEGylated zeinmicelles using a dialysis method, according to an embodiment.

FIG. 11 illustrates a transmission electron microphotograph (TEM) ofcurcumin-loaded PEG-Zein micelles positively stained with 1% w/v uranylacetate. Scale 1 mm=0.05 gm.

FIG. 12 illustrates atomic force microscopy (AFM) images ofcurcumin-loaded PEG-Zein micelles at scan rate of 2 μm in thenon-tapping mode. Left to right are 2D topography, amplitude, and phaseimages of a representative sample with z-scale of 88 nm, 0.39 V, and61°, respectively. The average particle size of 100 particles measuredin AFM was 90±10 nm.

FIG. 13 illustrates a UV-Visible spectrum of curcumin (10 μg/mL) inmethanol, PBS pH 7.4 (with 10% methanol) and curcumin-loaded PEGylatedzein micelles in PBS pH 7.4. The absorbance of curcumin-loaded PEG-Zeinis similar to the absorbance of curcumin solubilized in methanol,showing the increased water solubility of curcumin loaded PEG-zeinmicelles.

FIG. 14 illustrates the fluorescence spectra of curcumin (10 μg/mL) inmethanol, PBS pH 7.4 (with 10% methanol) and curcumin-loaded PEGylatedzein micelles in PBS pH 7.4. The shift of the λ_(max) of the emissionspectra of curcumin from 540 nm to 525 nm shows that the curcumin isentrapped in the micelles. There is also a significant increase(approximately 4 fold) in curcumin fluorescence in water afterentrapment in PEGylated zein micelles due to the significantly enhancedaqueous solubility of curcumin.

FIG. 15 illustrates differential scanning calorimetry (DSC) thermogramsof zein, curcumin, blank PEG-Zein, mPEG-ester, and curcumin-loadedPEGylated zein micelles.

FIG. 16 illustrates an in vitro release profile of curcumin fromPEG-Zein micelles in citrate buffer pH 7.4 (average±SE; n=3). Curcuminloaded PEG-Zein micelles (1 mg/mL) were prepared by a dialysis method asdescribed herein, were incubated in 1 mL of citrate buffer pH 7.4 in acentrifuge tube, and the suspension was maintained at 37° C. in ahorizontal shaker water bath at 50 rpm. The sample was centrifuged at12,000 rpm for 12 minutes. The supernatant was then analyzed forcurcumin released from the PEG-Zein micelles using HPLC. A C18 column(WATERS™ Corporation, MA, USA) was used and the mobile phase consistedof 60% acetonitrile and 40% citric buffer (1% (w/v) citric acid solutionadjusted to pH 3.0 using 50% (w/w) sodium hydroxide solution). The flowrate was 1.0 mL/min and the detection wavelength was 420 nm. The releasestudy was conducted for 24 hours. Each data point is a mean of threeexperiments±SD.

FIG. 17 illustrates an in vitro cytotoxicity profile of curcumin(dissolved in 10% DMSO) and curcumin micelles. NCl/ADR-RES drugresistant human ovarian cancer cells (2000 cells per well) that are drugresistant were treated with curcumin solution or curcumin micelles inthe concentration range of 7.8 nM to 500 nM for 4 days. On the fifth daycytotoxicity analysis was performed using an MTT assay. Data pointsrepresent average±SE (n=4). The IC₅₀ value for curcumin solution(Curcu-soln) and curcumin micelles (Curcu-M) was 104 nM and 34 nM,respectively.

FIG. 18 illustrates the in vitro skin penetration of free curcumin (10%TWEEN 80 in PBS, pH 7.4; represented as “C” in the figure) andencapsulated curcumin (in PBS, pH 7.4; represented as “CM” in thefigure) using excised porcine skin after different periods of treatmentin a vertical diffusion cell. Excised porcine skin was sandwichedbetween the two compartments of a vertical diffusion cell. The receptormedium consisted of phosphate buffer (pH 7.4 with 20% ethanol)maintained at 37° C. and stirred using a magnetic bead. The skin waswashed and tape-stripped 15-20 times using SCOTCH TAPE to remove stratumcorneum (SC). The curcumin was extracted from the tape strips and theremaining skin (viable epidermis+dermis) using 90% ethanol. The amountof curcumin in the skin and in the receptor phase was determined by HPLCmethod.

FIG. 19 illustrates the confocal fluorescence XZ optical scan images(0-100 μm depth) of porcine skin after 6 hours of treatment with freecurcumin (a) and curcumin encapsulated in PEG-zein micelles (b) andpenetration of curcumin micelles (c) through hair follicles (xy surfaceview) and (d) curcumin fluorescence pixels quantified in the stratumcorneum (SC) and viable epidermis. For SC 0-20 μm and for epidermis20-100 μm XZ optical sections were used for quantifying the fluorescencepixels.

FIG. 20 illustrates steps for the preparation of doxorubicin-loadedPEGylated zein micelles using a film method, according to an embodiment.

FIG. 21 illustrates steps for the preparation of doxorubicin-loadedPEGylated zein micelles using a dialysis method, according to anembodiment.

FIG. 22 illustrates a transmission electron microphotograph (TEM) ofdoxorubicin-loaded PEG-Zein micelles positively stained with 1% w/vuranyl acetate. Scale 1 mm=0.1 1 μm.

FIG. 23 illustrates atomic force microscopy (AFM) images ofdoxorubicin-loaded PEG-Zein micelles at scan rate of 2 μm in thenon-tapping mode. Left to right are 2D topography, amplitude, and phaseimages of a representative sample with z-scale of 228 nm, 0.64 V, and71°. The average particle size of 100 particles is 125±15 nm.

FIG. 24 illustrates the UV-Visible spectra of doxorubicin (10 μg/mL) inphosphate buffer pH 7.4, doxorubicin in 90% ethanol, and doxorubicinloaded PEGylated zein micelles in PBS pH 7.4, respectively. Theabsorbance of the doxorubicin-loaded PEG-Zein is higher than theabsorbance of doxorubicin solubilized in 90% v/v ethanol, due to theenhanced aqueous solubility of doxorubicin in PEG zein micelles.

FIG. 25 illustrates a fluorescence spectra of doxorubicin (10 μg/mL) inphosphate buffer pH 7.4, 90% ethanol, and doxorubicin-loaded PEGylatedzein micelles in PBS pH 7.4, respectively. There is a significantincrease (approximately 50 fold) in doxorubicin fluorescence in PBS pH7.4 after entrapment in PEGylated zein micelles due to the significantlyenhanced aqueous solubility of doxorubicin.

FIG. 26 illustrates differential scanning calorimetry (DSC) thermogramsof zein, doxorubicin, blank PEG-Zein, mPEG-ester, and doxorubicin-loadedPEGylated zein micelles.

FIG. 27 illustrates an in vitro release profile of doxorubicin fromPEG-Zein micelles in a citrate buffer pH 7.4 (n=3, ±SEM).Doxorubicin-loaded PEG-Zein micelles (1 mg/mL) were incubated in 1 mL ofthe citrate buffer pH 7.4 in a centrifuge tube and the suspension wasmaintained at 37° C. in a horizontal shaker water bath at 50 rpm. Thesample was centrifuged at 12,000 rpm for 12 minutes. The supernatant wasanalyzed for doxorubicin released from the PEG-Zein micelles using HPLCanalysis. A C18 column (WATERS™ Corporation, MA, USA) was used and themobile phase consisted of trifluoroacetic acid (0.1% v/v) andacetonitrile 5% v/v (˜3 min, 80% v/v ˜11 min and 5% v/v ˜22 minutes) ata flow rate of 1 mL/min. A fluorescence detector was used (505 nm as theexcitation and 550 nm as the emission wavelengths, respectively). Therelease study was conducted for 24 hours. Each data point is a mean ofthree experiments±SD.

FIG. 28 illustrates an in vitro cytotoxicity profile of doxorubicin base(dissolved in 90% v/v ethanol) and PEG-Zein micelles. MCF-7 human breastcancer cells (2000 cells per well) were treated with doxorubicinsolution (Dox-soln) and doxorubicin loaded micelles (Dox M) at aconcentration range of 15.62 nM to 1000 nM for 4 days. On day 5cytotoxicity analysis was performed using an MTT assay. Data pointsrepresents average±SE (n=4). The IC₅₀ values of doxorubicin anddoxorubicin micelles was 148 nM and 30 nM, respectively.

FIG. 29 illustrates an in vitro cytotoxicity profile of doxorubicin base(dissolved in 90% v/v ethanol) and doxorubicin loaded PEG-Zein micelles.NCl/ADR-RES drug resistant human ovarian cancer cells (2000 cells perwell) that are drug resistant were treated with doxorubicin solution(Dox-soln) or doxorubicin micelles (Dox-M) in the concentration range of31.25 nM to 1000 nM for 4 days. On day 5 cytotoxicity analysis wasmeasured using an MTT assay. Data points represent average±SE (n=4). TheIC₅₀ values for the doxorubicin and doxorubicin micelles were 126 nM and29 nM, respectively.

FIG. 30 illustrates the influence of temperature on cellular uptake ofdoxorubicin loaded PEG-Zein micelles in an NCl/ADR-RES cell line. Cellswere pre-incubated at 4° C. for 2 hours. After 2 hours cells were washedtwice with PBS pH 7.4, treated with the doxorubicin loaded PEG-Zeinmicelles (corresponding to 5 μg/mL of doxorubicin). After two hours, thetreatment was removed and cells were washed twice with ice cold PBS pH7.4, and the amount of doxorubicin content in the cell lysate wasestimated using HPLC analysis. In the control group, cells wereincubated at 37° C. Each value represents average±SE (n=3). The celluptake at 4° C. was significantly lower compared the cell uptake at 37°C. (p<0.05; student t-test).

FIG. 31 illustrates the kinetics of cellular uptake ofdoxorubicin-loaded PEG-Zein micelles and doxorubicin solution (51Lig/mL) in NCl/ADR-RES cell line (50000 cells/plate). Data pointsrepresent mean of three experiments±SE.

FIG. 32 illustrates the mechanism of cell uptake of PEG-zein micelles inresistant human cancer cells. NCl/ADR-RES cells (5000 cells/well) weretreated PLURONIC F68 (1 mg/mL; positive control; a block-copolymer knownto inhibit P-glycoprotein (P-gp)), and blank PEG-Zein micelles (0.050mg/mL). After 30 minutes of incubation at 37° C., 50 μL of 0.25 μM/L ofcalcein AM was added. Fluorescence was measured every 5 minutes for 1hour using a micro plate reader (485/589 Excitation/Emissionwavelengths) at room temperature. P-gp inhibition was calculated asfollows: % relativefluorescence=100×(FL_(treatment)−FL_(Non-treatment))/FL_(Non-treatment)).Data points represent average±SE (n=8). A higher P-gp inhibition wasobserved with blank PEG-Zein micelles.

FIG. 33 illustrates in vivo biodistribution of doxorubicin solution anddoxorubicin loaded PEG-Zein micelles (in saline) in mice allograftbreast tumor mouse model. The tumor model was developed by subcutaneousinjection of JC mouse breast cancer cells. Doxorubicin solution ormicelles were given by tail vein injection (4.5 mg/kg). Animals weresacrificed 3 hours after treatment administration. Tumor and organs werecollected. Doxorubicin concentration in tumors and organs weredetermined using a fluorescence based isocratic HPLC method. Doxorubicincontent was normalized to the organ weight (n=3-4, ±SEM). Micellesresulted in higher distribution to the tumor and significantly lowerdistribution in other organs. Doxorubicin is known to causecardiotoxicity and renal toxicity. The results show that micelles leadto enhanced efficacy and reduced toxicity of doxorubicin.

FIG. 34 illustrates in vivo anticancer efficacy of doxorubicin solutionand doxorubicin loaded PEG-zein micelles in drug resistant tumorallograft mouse tumor model. Female BALB/C bearing subcutaneous JC mousebreast cancer cells were used for the study. The mice were injected withdoxorubicin solution or micelles by i.v. injection on days 0 and day 7(3 mg/kg). Tumor volume was measured on alternate days. Percentreduction in tumor volume was calculated using the equation (tumorvolume after treatment/tumor volume before treatment)×100. Data isrepresented as mean±SEM, n=4-5 per group; * indicates the value issignificant at p<0.05 compared to other treatments. Except for thedoxorubicin micelles group, the mice did not survive after 7 days in allthe other treatment groups. Tumors grew slowly when doxorubicin micelleswere administered, signifying the greater efficacy of the micelleformulation. BM refers to blank micelles; Dox-Soln refers to adoxorubicin solution; and Dox-M refers to doxorubicin loaded PEG-Zeinmicelles.

FIG. 35 illustrates Kaplan-Meier survival plot of BALB/C mice bearingallogenic breast tumors. Female BALB/C bearing subcutaneous JC tumorswere injected with doxorubicin solution or doxorubicin micelles by i.v.injection (6 mg/kg), on day 0 and 7 in divided doses. Percent survivalof animals was plotted using the Graph Pad 5 software. Data is mean of4-5 animals per group. Mortality rate of mice were in the increasingorder of Dox micelles (Dox-M)<Dox-solution (Dox-Soln)<saline<blankmicelles (BM). The data shows that doxorubicin micelles resulted ingreater survival due to enhanced efficacy of the formulation.

FIG. 36 illustrates steps for the preparation of Nile red-loadedPEGylated zein micelles using a dialysis method, according to anembodiment.

FIG. 37 illustrates the quantity of Nile red in the epidermis andstratum corneum (n=3). An in-vitro study was carried out usingdermatomed porcine skin sandwiched between the two compartments of avertical diffusion cell (PERMEGEAR™, Hellertown, Pa.). 100 μL of Nilered (250 ng) in 5% v/v Tween-80 solution or Nile red-loaded PEG-Zeinmicelles in water (250 ng) was added to the donor compartment. Thereceptor compartment consisted of PBS pH 7.4 maintained at 37° C. andstirred with a magnetic stir bar. After 6 hours the skin was removed andthe fluorescence pixels were measured using confocal laser scanningmicroscopy (FLUOVIEW FV300™, Olympus ix70, Olympus, Center Valley, Pa.).Optical sections (xyz) were analyzed for fluorescence intensity in theStratum Corneum (0-15 μm) and viable epidermis (20-100 μm) usingFLUOVIEW™ software (Olympus, Center Valley, Pa.).

FIG. 38 illustrates by means of a flow chart the general steps toprepare retinol loaded PEGylated zein micelles using a dialysis method,according to one embodiment. In FIGS. 38-39 and 43-46, BHT refers tobutylated hydroxyltoluene (2,6-di-tert-butyl-4-methylphenol).

FIG. 39 illustrates by means of a flow chart the general steps toprepare retinol loaded PEGylated zein micelles using a film method,according to one embodiment.

FIG. 40 illustrates the water dispersibility of free retinol and retinolloaded nanomicelles, from left to right.

FIG. 41 illustrates the in vitro release of retinol from PEG-zeinnanomicelles in phosphate buffer (pH 7.4). The retinol concentration wasmeasured by UV-visible spectrophotometry at 320 nm. Each data point isan average±SD (n=3) (mean±SEM; n=3).

FIG. 42 illustrates free retinol, lyophilized and retinol micelles, fromleft to right. The figure shows the hygroscopic nature of pure retinoland that the retinol micelles are non-hygroscopic free flowing powders.

FIG. 43 illustrates the solid state stability of retinol loadednanomicelles when stored under normal room light. Free retinol andretinol micelles were kept in a clear glass vials and exposed to roomlight for one week. The retinol remaining at different time points wasmeasured by UV-visible spectrophotometry at 320 nm (mean±SD; n=3).

FIG. 44 illustrates the solid state stability of retinol loadednanomicelles when stored when stored in the absence of light. Freeretinol and retinol micelles were kept in a clear glass vials and storedin a dark cabinet for one week. The retinol remaining at different timepoints was measured by UV-visible spectrophotometry at 320 nm (mean±SD;n=3).

FIG. 45 illustrates the liquid state stability of retinol loadednanomicelles when stored under normal light. Free retinol and retinolmicelles were dispersed in phosphate buffer (pH 7.4) and stored in aclear glass vials in room light for one week. The retinol remaining atdifferent time points was measured by UV-visible spectrophotometry at320 nm (mean±SD; n=3).

FIG. 46 illustrates the liquid state stability of retinol loadednanomicelles when stored protected from light in dark cabinet. Freeretinol and retinol micelles were dispersed in phosphate buffer (pH 7.4)and stored in a clear glass vials in a dark cabinet for one week. Theretinol remaining at different time points was measured by UV-visiblespectrophotometry at 320 nm (mean±SD; n=3).

FIG. 47 illustrates the percentage of applied retinol at the end of 48hours in porcine skin and in receptor medium after treatment with freeretinol and retinol encapsulated in PEG-zein micelles. Excised porcineskin was sandwiched between the two compartments of a vertical diffusioncell. The receptor medium consisted of phosphate buffer (pH 7.4)maintained at 37° C. and stirred using a magnetic bead. Free orencapsulated retinol dispersion in phosphate buffer (pH 7.4) was loadedin the donor chamber. At the end of the study, the retinol concentrationin the skin and receptor compartment was measured by radiochemicalmethod using ³H labeled retinol. The skin was digested using 0.1M sodiumhydroxide to determine the retinol concentration (mean±SD; n=6).

FIG. 48 illustrates the percentage of applied retinol at the end of 48hours in porcine skin and in receptor medium after treatment with freeretinol and retinol encapsulated micelles. Excised porcine epidermis(Epi) was placed between the two compartments of a vertical diffusioncell. In the second set of experiments, the stratum corneum was removedfrom the porcine epidermis and then was physically placed (sandwiched)over the porcine epidermis (Sand) and was used in the study. Freeretinol or retinol nanomicelles were applied over the skin and the studywas conducted for 48 hours. The receptor medium consisted of phosphatebuffer (pH 7.4) maintained at 37° C. and stirred using a magnetic bead.Free or encapsulated retinol dispersion in phosphate buffer (pH 7.4) wasloaded in the donor chamber. At the end of the study, the retinolconcentration in the skin and receptor compartment was measured byradiochemical method using ³H labeled retinol. The skin was digestedusing 0.1M sodium hydroxide to determine the retinol concentration(mean±SD; n=6).

FIG. 49 illustrates the stability of the retinol micelle creamformulation stored at room temperature and 49° C. for a period of onemonth in glass vials covered with aluminum foil. At regular intervals analiquot of the formulation was removed and the retinol content wasanalyzed using HPLC. The formulation remained stable and did not showany significant degradation at room temperature. Each vial is a mean±SD;n=3.

FIG. 50 illustrates in vitro release of free retinol (filled circle) andretinol micelles (filled triangle) from cream formulation at pH 7.4.

FIG. 51 illustrates the in vitro skin penetration of retinol creamformulations in human skin.

FIG. 52 illustrates the transepidermal water loss (TEWL) values in miceafter application of free and micelle encapsulated cream retinolformulations. Sodium lauryl sulfate (SLS), a known skin irritant, wasused as the positive control, and the negative control group was notsubjected to any treatment.

FIG. 53 illustrates the in vivo topical bioavailability of free andnanoparticle encapsulated retinol after treatment for 6 hours in SKH-1hairless mice.

FIG. 54 illustrates the steps to prepare Nile red loaded caseinmicelles.

FIG. 55 illustrates the fluorescence pixels in the different layers ofthe skin after 6 hours of treatment with free Nile red and Nile redencapsulated in casein micelles. For stratum corneum (SC), 0-20 μm andfor epidermis 20-100 μm XZ optical sections were used for quantifyingthe fluorescence pixels.

DETAILED DESCRIPTION OF THE INVENTION

Zein, a hydrophobic plant protein, belongs to a family of prolamines andis water insoluble. Zein has been investigated as a polymer forsustained release of various agents in the pharmaceutical, food, andcosmetic industries (Shukla and Cheryan (2001), Ind Crops Prod13:171-192). Zein has also been used to film coat materials and to formparticulate systems such as microparticles or nanoparticles.Polyethylene glycol (PEG) is a water soluble, biocompatible FDA approvedpolymer composed of multiple ethylene glycol units linked by etherbonds.

Applicants have discovered that various amphiphilic protein conjugatescan self-assemble to form stable, biocompatible, and biodegradablemicellar assemblies, as schematically as illustrated in FIG. 1. Themicelles can be formed with or without cargo molecules in the micellecore. It was also discovered that zein can be covalently attached topolyethylene glycol (PEG) as described in FIGS. 2 and 3. Blank (non-drugloaded) or drug loaded PEGylated zein self-assembles in an aqueousenvironment to form nanomicelles (−100 nm) with a hydrophobic core and ahydrophilic shell.

Other hydrophobic proteins can be used in place of zein, for example,those derived from a variety of sources including plants, animals andsynthetic sources. Similarly, other water soluble polymers such aspolyvinylpyrrolidone, polyglycolic acid, and others described herein canbe conjugated to the hydrophobic proteins to prepare the nanomicelles.Various water insoluble hydrophobic molecules (e.g., therapeutic agentsor “drugs”) can be encapsulated inside the core of the nanomicelle, andthe hydrophilic polymeric chains at the corona of the micelle help tosolubilize the drug in an aqueous environment, such as the human body.Additionally, charged molecules neutralized with counter ions can beencapsulated inside the hydrophobic core of a micelle described herein.Alternatively, when charged functional groups are introduced into thehydrophobic core or hydrophilic shell, the charged molecules can becomplexed to the core and/or to the shell through hydrostaticinteractions. For example, attachment of cationic polymers, such aspolyethylene imine, polylysine, and the like, to the micelle core and/orshell can be used to complex negatively charged DNA or oligonucleotides.Similarly, hydrophilic molecules can be chemically modified (e.g., intothe form of a prodrug or salt) to provide a hydrophobic entity forencapsulation in the core of a micelle. In embodiments, the overallcharge on the protein may changed by adjusting pH above or below the pIof the prolamine (e.g., pI of zein is between about 5 and 9; pI ofgliadin is about 6.8)

DEFINITIONS

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14th Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The terms “comprising,” “including,” “having,” “containing,”“characterized by,” and grammatical equivalents thereof, are inclusiveor open-ended terms that do not exclude additional, unrecited elementsor method steps, but also include the more restrictive terms “consistingof and “consisting essentially of’.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” (e.g., a drug) includes a plurality of such compounds,so that a compound X includes a plurality of compounds X. As anadditional example, reference to “a micelle” can include a plurality ofsuch micelles, and reference to a “molecule” is a reference to aplurality of molecules, and equivalents thereof. It is further notedthat the claims may be drafted to exclude any optional element. As such,this statement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely”, “only”, and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer. Unless otherwise indicated herein, the term“about” is intended to include values, e.g., weight percents, proximateto the recited range that are equivalent in terms of the functionalityof the individual ingredient, the composition, or the embodiment. Inaddition, unless indicated otherwise herein, a recited range (e.g.,weight percents or carbon groups) includes each specific value oridentity within the range.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible subranges andcombinations of subranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc.

As will also be understood by one skilled in the art, all language suchas “up to,” “at least,” “greater than,” “less than,” “more than,” “ormore,” and the like, include the number recited and such terms refer toranges that can be subsequently broken down into subranges as discussedabove. In the same manner, all ratios recited herein also include allsubratios falling within the broader ratio. Accordingly, specific valuesrecited for radicals, substituents, and ranges, are for illustrationonly; they do not exclude other defined values or other values withindefined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “zein” refers to a class of prolamine protein. Prolamines arefound in various grains such as corn, wheat, barley, rice, and sorghum,as well as in other plants and animals. Other examples of prolaminesinclude gliadin, hordein and kafirin. These prolamines can be exchangedfor zein in the various embodiments described herein. Zein is composedof a high proportion of non-polar amino acids, such as proline,glutamine and asparagine, and has a molecular weight of about 22-27 kDa(Shukla, Zein: the industrial protein from corn. Ind. Crops. Prod. 13,171-92; 2001). A typical sample of zein can have approximately 20%leucine, 10% proline, 21-26% glutamine, 5% asparagine, and 10% alanine,therefore at least about 61% of its amino acid composition is ofhydrophobic amino acids. These hydrophobic amino acids render theprotein water insoluble. Zein is a biodegradable US-FDA approved GRASpolymer (Fed. Register (1985) 50:8997-8999).

Zein can be manufactured as a powder from corn gluten meal. Pure zein isodorless, tasteless, water-insoluble, and edible, properties which haverendered it an important component for processed foods andpharmaceuticals. Methods for isolating, processing, and using zein arewell known in the art. See for example, Lawton, Cereal Chem 2002, 79(1):1-18, and WO 2009/137112 (Perumal et al.), which are incorporated hereinby reference. A “grade” of zein refers to a variety of types or forms ofzein, including white zein and yellow zein, derived by various means,such as is disclosed in U.S. Pat. No. 5,254,673 (Cook et al.), thecontents of which are incorporated by reference herein.

The term “PEG” or “polyethylene glycol” refers to a water soluble,biocompatible FDA approved polymer composed of multiple ethylene glycolunits linked by an ether bond. The molecular weight of a PEG chain ormoiety can vary from about 1 kDa to about 220 kDa, for example, about 1kDa to about 15 kDa, depending on the number of ethylene glycol units inthe chain. PEG moieties can be represented as —(OCH₂CH₂)_(n)OH or—(OCH₂CH₂)_(n)OR groups where n is 2 to about 1,000 and R is alkyl,aryl, or arylalkyl such as methyl, ethyl, t-butyl, phenyl, or benzyl.PEG moieties can be attached to proteins through the terminal hydroxylgroup, for example, when activated with succinate esters.

In various embodiments, the molecular weight of the PEG chain can beabout 1 kDa to about 220 kDa. In certain embodiments, the PEG group canhave a molecular weight of about 1,000 to about 20,000, about 4,500 toabout 20,000; about 5,000 to about 18,000; about 5,000 to 20 about12,000; or about 4,000 to about 9,000. In other embodiments, the PEGgroups can have a molecular weight of about 4,000, 5,000, 6,000 or about7,000. The PEG group can also be capped at its terminal end with aprotecting group, such as an acetyl group or an alkyl group, forexample, a methyl or an ethyl group.

Heterobifunctional PEG groups, which have dissimilar terminal groups,can also be used for PEGylation. Examples of heterobifunctional PEGgroups include HO₂C-PEG-OH; HC(═O)—PEG-SH, and the like. In addition tolinear PEG moieties, branched moieties that include PEG chains can alsobe used for PEGylation of a prolamine Examples of various PEG moietiesthat can be conjugated to zein are described by Roberts et al. (Adv.Drug Deliv. Rev. 54:459-476, 2002) and are illustrated below.

Branched PEG groups based on PEG2 triazine:

where the amino group of R—NH2 is an amino group on a side chain orterminal group of a prolamine protein. Other PEG moieties that can beconjugated to a prolamine protein include: 1) Branched PEG (PEG2);

2) linear forked PEG; and/or

3) branched forked PEG:

where Y is a group having a carbon branching moiety and X is an atom ofa prolamine protein, a linker to a prolamine protein, or a functionalgroup of a prolamine protein.

Polyethylene glycol moieties or other poly(alkylene oxides) can beconjugated to zein by a variety of techniques well known in the art (seefor example, Francesco et al. (2005), Drug Discov Today 10, 1451-1458).One example of conjugate formation includes reacting a prolamine proteinsuch as zein with an activated monoalkoxylated PEG ester, such asmethoxy PEG-succinimidyl succinate, to form ester or amide linkages, asillustrated in Scheme A below.

As shown in Scheme A above, m-PEG-N-hydroxy succinimidyl ester can beconjugated through formation of an amide bond to one or more terminalamine groups of glutamine residues (and/or asparagine residues) in aprolamine, such as zein (Sessa et al., (2007) J Appl Poly Sci 105,2877-2883). In other embodiments, the amine groups in arginine andhistidine can be conjugated to PEG through an amide or carbamatelinkage. In addition, the N-terminus amino acids can be PEGylated.Various PEG derivatives known in the art can be used for PEGylating theamine groups, including PEG caryboxylic acids, esters, carbonates,aldehydes, and the like. Carboxylic acids in aspartic acid and glutamicacid, as well as the C-terminal carboxylic acids in zein, can also beconjugated to PEG using PEG with amine, hydroxyl, or other functionalgroups known in the art for linking carboxylic acids to PEG groups. Thethiol in cysteine in zein can also be conjugated to PEG using, forexample, PEG functionalized with pydriyl sulfide, vinyl sulfone,maleimide, or iodoacetamide. Threonine and serine in zein can also bePEGylated, using techniques well known in the art.

Site specific PEGylation in zein can be achieved using enzymes. Forexample, transglutaminase can be used to selectively PEGylate the sidechain amine group in glutamine as shown in Scheme B below. Similarlyselective PEGylation can be achieved by selective glycosylation ofhydroxyl group in serine or threonine using acetylgalactosylaminetransferase followed by conjugation of PEG-sialic acid usingsialyltransferase (Veronese et al. (2005), Drug Discov Today 10,1451-1458).

In various embodiments, other alkylene oxides can be used in place ofpolyethylene glycol, such as alkylene oxide chains that contain from 2to 4 carbon atoms in each alkylene group. Alkoxy-terminatedpoly(alkylene oxides) are suitable examples, such as methoxy-terminatedpoly(alkylene oxides), and the free hydroxy end can then be activatedwith groups such as succinimidyl succinates. In some embodiments, thepoly(alkylene oxide) chains can have from about 2 to about 110 repeatingunits, and typically have from about 50 to about 110 repeating units.

The term “biocompatible” means that the polymer or conjugate referred todoes not cause or elicit significant adverse effects when administeredin vivo to a subject. Examples of possible adverse effects include, butare not limited to, excessive inflammation and/or an excessive oradverse immune response, as well as toxicity. Zein and polyethyleneglycol are biocompatible components.

The term “hydroalcoholic solvent” refers to a solvent system thatincludes both water and an alcoholic solvent, such as methanol, ethanol,n-propanol, iso-propanol, or butanol (including 1-butanol, 2-butanol(sec-butanol), iso-butanol, and tert-butanol). Common hydroalcoholicsolvent systems include 50%, 70%, 90%, and 92% ethanol in water.

The term “stable” refers to a core of a micelle where the core has nocontact with water (see, e.g., Core-Shell structure of PEG-Zein, Example1).

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

The term “administered” or “administration” when used in the context oftherapeutic and diagnostic uses for micelles, refers to and includes theintroduction of a selected amount of micelles into an in vivo or invitro environment for the purpose of, for example, delivering atherapeutic agent to a targeted site.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an amount effective can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a blank or drug loaded micelledescribed herein, e.g., that is effective to treat or prevent a diseaseor disorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect.

The terms “treating”, “treat” and “treatment” can include (i) preventinga disease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can includes both medical,therapeutic, and/or prophylactic administration, as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “in vivo” means of or within the body of a subject, such asthat of a patient, and includes administration of micelles by a varietyof means including, but not limited to, oral, buccal, intravenous,intramuscular, intraperitoneal, parenteral, subcutaneous, topical,ocular, pulmonary and nasal routes of administration.

The term “in vitro” refers to environments outside of the body of asubject or patient. The terms “subject” or “patient” both refer to anindividual complex organism, e.g., a human or non-human animal.

The term “therapeutic agent,” and similar terms referring to atherapeutic or medicinal function mean that the referenced smallmolecule, macromolecule, protein, nucleic acid, growth factor, hormone,drug, other substance, cell, or combination thereof can beneficiallyaffect the initiation, course, and/or one or more symptoms of a diseaseor condition in a subject, and may be used in conjunction with micellesin the manufacture of medicaments for treating a disease or othercondition. Suitable therapeutic agents for encapsulation in the micellesdescribed herein include hydrophobic therapeutic agents, for example,but not limited to, curcumin, doxorubicin, and imaging agents such asNile red.

Hydrophobic Agents:

Practically any hydrophobic agent otherwise suitable for the practice ofthis invention may be employed for a variety of applications. Theamphiphilic polymers described herein may also be used as thickeningagents, lubricants, detergents, surfactants, and anti-fouling agents.The amphiphilic polymers may be used as an emulsifying, dispersing orstabilizing agent for dyes, cosmetics, pigment and pharmaceuticalproducts. The amphiphilic polymers can be particularly useful as anemulsifying, dispersing or stabilizing agent in the dyeing of textilesand for encapsulating dyes for cosmetics. The amphiphilic polymers canbe useful as lubricants and encapsulants for cosmetics, pharmaceuticals,nutraceuticals, pesticides, textiles, and perfumes.

Thus, in addition to biologically or pharmaceutically active hydrophobicagents, other hydrophobic molecules that may be encapsulated by theamphiphilic polymers described herein include diagnostic agents,insecticides, pesticides, herbicides, antiseptics, food additives,fragrances, dyes, diagnostic aids, and the like. Examples of hydrophobicmolecules that can be encapsulated by the amphiphilic polymers describedherein include, but are not limited to: abietic acid, aceglatone,acenaphthene, acenocoumarol, acetohexamide, acetomeroctol, acetoxolone,acetyldigitoxins, acetylene dibromide, acetylene dichloride,acetylsalicylic acid, alantolactone, aldrin, alexitol sodium, allethrin,allylestrenol, allyl sulfide, alprazolam, aluminumbis(acetylsalicylate), ambucetamide, aminochlothenoxazin,aminoglutethimide, amyl chloride, androstenediol, anethole trithone,anilazine, anthralin, Antimycin A, aplasmomycin, arsenoacetic acid,asiaticoside, astemizole, aurodox, aurothioglycanide, 8-azaguanine,azobenzene; baicalein, Balsam Peru, Balsam Tolu, barban, baxtrobin,bendazac, bendazol, bendroflumethiazide, benomyl, benzathine,benzestrol, benzodepa, benzoxiquinone, benzphetamine, benzthiazide,benzyl benzoate, benzyl cinnamate, bibrocathol, bifenox, binapacryl,bioresmethrin, bisabolol, bisacodyl, bis(chlorophenoxy)methane, bismuthiodosubgallate, bismuth subgallate, bismuth tannate, Bisphenol A,bithionol, bornyl, bromoisovalerate, bornyl chloride, bornylisovalerate, bornyl salicylate, brodifacoum, bromethalin,broxyquinoline, bufexamac, butamirate, butethal, buthiobate, butylatedhydroxyanisole, butylated hydroxytoluene; calcium iodostearate, calciumsaccharate, calcium stearate, capobenic acid, captan, carbamazepine,carbocloral, carbophenothin, carboquone, carotene, carvacrol,cephaeline, cephalin, chaulmoogric acid, chenodiol, chitin, chlordane,chlorfenac, chlorfenethol, chlorothalonil, chlorotrianisene,chlorprothixene, chlorquinaldol, chromonar, cilostazol, cinchonidine,citral, clinofibrate, clofazimine, clofibrate, cloflucarban, clonitrate,clopidol, clorindione, cloxazolam, coroxon, corticosterone, coumachlor,coumaphos, coumithoate cresyl acetate, crimidine, crufomate, cuprobam,cyamemazine, cyclandelate, cyclarbamate cymarin, cypernethril; dapsone,defosfamide, deltamethrin, deoxycorticocosterone acetate,desoximetasone, dextromoramide, diacetazoto, dialifor, diathymosulfone,decapthon, dichlofluani, dichlorophen, dichlorphenamide, dicofol,dicryl, dicumarol, dienestrol, diethylstilbestrol, difenamizole,dihydrocodeinone enol acetate, dihydroergotamine, dihydromorphine,dihydrotachysterol, dimestrol, dimethisterone, dioxathion, diphenane,N-(1,2-diphenylethyl)nicotinamide, dipyrocetyl, disulfamide, dithianone,doxenitoin, drazoxolon, durapatite, edifenphos, emodin, enfenamic acid,erbon, ergocorninine, erythrityl tetranitrate, erythromycin stearate,estriol, ethaverine, ethisterone, ethyl biscoumacetate,ethylhydrocupreine, ethyl menthane carboxamide, eugenol, euprocin,exalamide; febarbamate, fenalamide, fenbendazole, fenipentol,fenitrothion, fenofibrate, fenquizone, fenthion, feprazone, flilpin,filixic acid, floctafenine, fluanisone, flumequine, fluocortin butyl,fluoxymesterone, fluorothyl, flutazolamn, fumagillin,5-furfuryl-5-isopropylbarbituric acid, fusafungine, glafenine, glucagon,glutethimide, glybuthiazole, griseofulvin, guaiacol carbonate, guaiacolphosphate, halcinonide, hematoprophyrin, hexachlorophene, hexestrol,hexetidine, hexobarbital, hydrochlorothiazide, hydrocodone, ibuproxam,idebenone, indomethacin, inositol niacinate, iobenzamic acid, iocetamicacid, iodipamide, iomeglamic acid, ipodate, isometheptene, isonoxin,2-isovalerylindane-1,3-dione; josamycin, 11-ketoprogesterone,laurocapram, 3-O-lauroylpyridoxol diacetate, lidocaine, lindane,linolenic acid, liothyronine, lucensomycin, mancozeb, mandelic acid,isoamyl ester, mazindol, mebendazole, mebhydroline, mebiquine,melarsoprol, melphalan, menadione, menthyl valerate, mephenoxalone,mephentermine, mephenyloin, meprylcaine, mestanolone, mestranol,mesulfen, metergoline, methallatal, methandriol, methaqualone,3-methylcholanthrene, methylphenidate, 17-methyltestosterone,metipranolol, minaprine, myoral, naftalofos, naftopidil, naphthalene,2-naphthyl lactate, 2-(2-naphthyloxy)ethanol, naphthyl salicylate,naproxen, nealbarbital, nemadectin, niclosamide, nicoclonate,nicomorphine, nifuroquine, nifuroxazide, nitracrine, nitromersol,nogalamycin, nordazepamn, norethandrolone, norgestrienone; octaverine,oleandrin, oleic acid, oxazepam, oxazolam, oxeladin, oxwthazaine,oxycodone, oxymesterone, oxyphenistan acetate, paraherquamide,parathion, pemoline, pentaerythritol tetranitrate, pentylphenol,perphenazine, phencarbamide, pheniramine, 2-phenyl 6-chlorophenol,phentlmethylbarbituric acid, phenyloin, phosalone,phthalylsulfathiazole, phylloquinone, picadex, pifarnine, piketopfen,piprozolin, pirozadil, plafibride, plaunotol, polaprezinc, polythiazide,probenecid, progesterone, promegestone, propanidid, propargite, propham,proquazone, protionamide, pyrimethamine, pyrimithate, pyrvinium pamoate;quercetin, quinbolone, quizalofo-ethyl, rafoxanide, rescinnamine,rociverine, runnel, salen, scarlet red, siccanin, simazine, simetride,sobuzoxane, solan, spironolactone, squalene, stanolone, sucralfate,sulfabenz, sulfaguanole, sulfasalazine, sulfoxide, sulpiride,suxibuzone, talbutal, terguide, testosterone, tetrabromocresol,tetrandrine, thiacetazone, thiocolchicine, thioctic acid, thioquinox,thioridazine, thiram, thymyl N-isoamylcarbamate, tioxidazole, tioxolone,tocopherol, tolciclate, tolnaftate, triclosan, triflusal, triparanol;ursolic acid, valinomycin, veraparnil, vinblastine, vitamin A, vitaminD, vitamin E, xenbucin, xylazine, zaltoprofen, and zearalenone.

A particular class of hydrophobic molecules having biological activitythat are suitable for use with the present invention are inter-cellularregulators and mediators such as interferons, growth factors, hormones,and the like, including their cognate receptors. The amphiphilicconjugates described herein are contemplated to be particularlyeffective for the efficient administration of interferons, which hasproven to be problematic because of interferon's water-insolubility.Topical dosage forms of the micellar formulations described herein canexhibit an unexpectedly accelerated rate of transdermal deliveryattributable to the encapsulation of the hydrophobic material by theamphiphilic polymer micelles. Thus, the polymer-encapsulated hydrophobicmaterial having biological or pharmaceutical activity may be prepared astopical dosage forms such as lotions, gels, salves, creams, balms,ointments and the like. These compositions may be in the form of aqueoussolutions, or in the form of oil-in-water or water-in-oil emulsions.These compositions can be formulated for administration to a patient bya variety of routes, including administration by injection, pulmonaryadministration, and administration by via oral or nasal routes. Theseformulations that include the micelles described herein can be otherwiseconventional formulations, optionally containing well-known additives,and can be prepared using art-recognized techniques.

Solubilization Technologies:

Numerous approaches have been used to solubilize hydrophobic drugs forimproving their delivery to patients. An overview of existingsolubilization technologies is illustrated below in Table A. The tableshows only a representative list of solubilization technologies used inmarketed products and in clinical development.

TABLE A Various Solubilization Technologies. Drug Delivery TechnologyExamples of Commercial Products Milling^(a): RAPAMUNE ® (Wyeth);NANOCRYSTAL ™ Technology EMEND ® (Apreipitant, MK869) (Merck); (ElanDrug Delivery) TRICOR ® (Fenofibrate) (Abott); MEGACE ® ES (Megestrol)(BMS); INVEGA ® SUSTENA ™ (Ortho McNeil Janssen) Modifiedcyclodextrins^(b): GEODON ® (Ziprasidone) (Pfizer); (Cydex Inc.)CAPTISOL ® VFEND ® (Voriconozole) (Pfizer); ABILIFY ® (Aripiprazole)(BMS); CORDARONE ® (Amiodarone) (Prism-Arrhythmia) Salts^(c) Amiodipinebesylate; Doxorubicin hydrochloride; Clopidogrel bisulphate Surfactantand polymeric micelles^(d): TAXOL ® - BMS Amphoteric: lecithin AQUASOLA ® Parenteral; AQUASOL E ® Drops; Non-ionic: Polysorbates (TWEEN,SPAN); Vitamin GENEXOL-PM - Phase-II clinical trials E-TPGS; CREMOPHOR ®EL; SOLUTOL ® HS 15; block co-polymers (e.g., PLURONICS ®); Ionic:Sodium lauryl sulfate (SLS); Self-emulsifying lipids (GELUCIRE, others)PEGylation of small molecule drugs^(e) PEG-docetaxel (NextarTherapeutics) - Phase I clinical trials; PEG-SN38 (EnzonPharmaceuticals) - Phase I clinical trials; PEG-irinotecan (NextarTherapeutics) - Phase II clinical trials ^(a)Lipinski (2002), Am. Pharm.Rev. 5: 82-85; Neervannan (2006), Expert Opin Drug Metab Toxicol 2:715-731. ^(b)Miller et al. (2006), J Pharm Sci, 96: 1691-1707; Redentiet al. (2000), J Pharm Sci 89: 1-8; Redenti et al. (2001), J Pharm Sci90: 979-986. ^(c)Yalkowsky et al. (1998), J Pharm Sci 87: 787-796;Portmann and Simmons (1995), J Pharm Biomed Analysis 13, 1189-1193;Johnson et al. (2003), J Pharm Sci 92: 1574-1581 ^(d)Torchillin (2007),Pharm Res 24: 1-16; Vries et al. (1996), Drug Dev Ind Pharm, 22:475-494; Tije et al. (2003), Clin Pharmacokinet 42: 665-685. ^(e)Pasutand Veronese (2009), Adv Drug Deliv Rev, 61, 1177-1188; Greenwald et al.(2000), Crit Rev Ther Drug Carrier Syst 17, 101-161; Veronese et al.(2005), Drug Discov Today 10, 1451-1458.

Overview of PEG Conjugates in Clinical Development or Use as AnticancerAgents:

Milling active agents (drugs) provides several advantages, includingscalability, low batch variability, and high flexibility in handlinglarge quantities of drugs. Disadvantages of milling active agents arethat the process may be applicable to only crystalline drugs, GRASlisted steric/ionic stabilizers may be needed, Ostwald ripening mayoccur, and prolonged milling may induce the formation of amorphouscompositions, leading to instability.

Modified cyclodextrins, such as β-cyclodextrin, is a GRAS and FDAapproved excipient. However, the cyclodextrins require a strictcorrelation between the structure of guest molecule and cavity size.Cyclodextrins can also significantly modify ADME parameters if thecorresponding binding constant is too high. Salts of active agents canbe used to provide improved aqueous solubility and can in some cases beused to advantageously alter a pharmacokinetic profile. Salts can alsoincrease the melting point of drug for processing. However, formation ofsalts requires suitable ionizable groups, and a salt of an active agentcan be considered a new drug by FDA and require separate approval. Saltscan also result in a common ion effect with hydrochloride salts, andhave a propensity for formation of hydrates and polymorphs, and/or alterpharmacokinetics.

Surfactant and/or polymeric micelles have less of a tendency toprecipitation on dilution, undesirable side effects are minimized, andhave been found to be a useful drug delivery system. However, manymicelle systems have various amounts of toxicity associated with theircomponent surfactants, loading capacity can be insufficient, andsolubilization capacity can be too low. Owing to their surface activity,surfactant molecules also have the potential to penetrate and disruptbiological membranes and can be hemolytic.

The critical micellar concentration (CMC) of micelles dictates theirstructural stability after in vivo administration, and polymericmicelles have higher structural stability than surfactant micelles.However, most of the polymeric micelles that are reported in theliterature are synthetic block copolymers that are prepared fromindividual monomeric units through tedious and complex syntheticprocedures.

PEGylation of small molecule drugs can be limited by the number offunctional groups in a drug. PEGylation of small drug molecules can alsocause conformational constraint and may affect the binding andtherapeutic activity of the drug. Furthermore, due to the limitedconjugation of PEG (i.e., one PEG per drug molecule) and limited drugloading with a polymer drug-conjugate, the increase in water solubilityof highly hydrophobic drugs is modest at best. Accordingly, improveddrug delivery systems are needed to overcome the many disadvantages ofcurrently used drug delivery technologies.

Micelles and Applications Thereof:

The present invention relates to a method of preparing micelles using ahydrophobic water insoluble protein and a water soluble polymer. Forexample, polyethylene glycol (PEG), a synthetic polymer, can becovalently conjugated to zein, a hydrophobic water insoluble plantprotein. Amphiphilic PEGylated zein can spontaneously formself-assembled micelles with a hydrophobic core and a hydrophilic shellwhen dispersed in water at a CMC of about 0.025 g/L. The diameter of themicelles can be, for example, about 10 nm to about 450 nm, about 10 nmto about 300 nm, about 75 nm to about 450 nm, about 75 nm to about 300nm, about 10 nm to about 200 nm, or about 80 nm to about 200 nm.

It was found that nanomicelles were formed only after covalentmodification of zein with polyethylene glycol of about 3 kDa or larger.PEG moieties of about 5 kDa were found to be especially suitable formicelle formation when conjugated to zein.

Because zein is a high molecular weight protein (approximately 22-27kDa), the PEGylated zein forms more stable micelles than most otherknown polymeric micelles. The concentration required for formation ofmicelles is known as critical micellar concentration (CMC). The CMCdetermines the stability of a micelle on dilution with water or serum.In this regard, the lower the CMC, the higher the stability of micelles.For example, sodium dodecyl sulfate has a CMC of about 2.304 g/L. TheCMC for PEGylated zein, in some embodiments, is 0.025±0.0095 g/L, whichis lower than the CMC value for commercially available block-copolymericmicelles prepared from polyethylene oxide and polypropylene oxide(PLURONIC®) polymers, which varies between 0.3 and 190 g/L, depending onthe molecular weight of PLURONIC® polymers.

PEG-zein micelles can be combined with other surfactant or polymers toform mixed micellar systems, for example, to enhance encapsulation orstability, or provide additional or varied functionality. Surfactantsthat can be used to form mixed micelles may include nonionic surfactantssuch as BRIJ 35, BRIJ 58P, TRITON X-100, TRITON X-114, TWEEN 20, TWEEN40, TWEEN 80, SPAN 80, and the like, or anionic surfactants such as bilesalts, sodium dodecyl sulfate, or cationic surfactants such ashexadecyltrimethyl ammonium bromide (CTAB), trimethyltetradecyl ammoniumbromide (TTAB), and the like.

PEG-prolamine graft copolymers or block-co-polymers can be used to formmixed micelles with other polymers that include PLURONICS (polypropyleneoxide-b-polyethylene oxide), polylactic acid-b-PEG,polycaprolactone-b-PEG, PEG-b-poly(N-isopropylacrylamide),PEG-b-poly(-(diethylamino)ethylmethacrylate)-b-poly(-(diethylamino)ethyl methacrylate),PEG-b-polyaminoacids, polyaspartic acid-b-PEG, PEG-b-polypropyleneoxide-b-polyethylene oxide, polylactic acid-b-polyethyleneoxide-b-polypropylene oxide, polyvinylpyrrolidone-b-polylacticacid-b-polyvinylpyrrolidone, poly((3-benzyl aspartate)-g-PEG,chitosan-g-polycaprolactone-g-PEG, and the like.

Similarly, lipids such as phospholipids, phosphatidylethanol amine,PEG-diacyllipids and the like can also be used to form mixed micelleswith the zein-PEG conjugates. Natural polymers such as casein can alsobe combined to form mixed micelles with PEG-zein. The surfactants,lipids, natural and synthetic polymers described above are onlyrepresentative examples and the composition of the surfactants, polymersor lipids in the micelles can be altered to form various mixed micelleswith PEG-zein.

Encapsulation of poorly soluble compounds into PEGylated zein micellescan be achieved by co-dissolving both components in a hydroalcoholicsolvent, such as 90% v/v ethanol, followed by incubation for an amountof time (e.g., overnight) sufficient to allow partitioning of thehydrophobic compound (“cargo molecule”) into the hydrophobic zein core.After incubation, the hydroalcoholic solvent can be removed byevaporation to form a thin film. The film can be reconstituted in abuffer to recover the drug loaded micelles.

Encapsulation of poorly soluble compounds into amphiphilic PEG-Zein canalso be achieved by co-dissolving both components in a hydroalcoholicsolvent (e.g., 90% v/v ethanol), followed by incubation to allowpartitioning of hydrophobic compound into hydrophobic zein core. Afterincubation, the hydroalcoholic solvent can be removed, for example, byextensive dialysis against water. Complete removal of the alcoholresults in formation of the drug loaded micelles.

Alternatively, a lyophilization method can also be used to preparePEG-zein micelles. The poorly soluble compound and PEG-zein can bedissolved in a water/tert-butanol solvent mixture followed by removal ofthe solvent by lyophilization. The micelles form spontaneously uponreconstitution of the freeze-dried product in an aqueous vehicle orbuffer.

PEGylated prolamine micelles have numerous important applications. Forexample, they can be used to enhance the solubility of hydrophobiccompounds of interest to the pharmaceutical and related industries, suchas those hydrophobic compounds with a Log P ranging from about 1 to 6.5(octanol/water) or greater. In some embodiments, an encapsulationefficiency of about 60% to about 95% can be achieved using the micellesdescribed herein. The micelles described herein can provide sustainedrelease of the encapsulated cargo molecules for up to about one week, orup to about two weeks, in an in vitro or in vivo environment.

The CMC, size and encapsulation efficiency of the micelles can also bevaried by changing the degree of PEGylation, molecular weight (m.w.) ofPEG moiety used, and the ratio of drug to polymer used in preparing themicelles. For example, the CMC can be lowered by using a highermolecular weight PEG. Similarly, the CMC can be reduced by optimizingthe number of PEG chains in a PEG-zein conjugate. A lower drug toPEG-zein ratio can also lead to smaller sized micelles. On the otherhand, an increase in drug/PEG-zein ratio can increase the encapsulationefficiency and loading efficiency in the micelles. Cross-linking thezein hydrophobic core or PEG shell can also increase the loadingefficiency. The cross-linking can also be used to further sustain therelease of cargo molecules from the micelles. Additionally, surfaceconjugation of targeting ligands can be used to specifically target themicelles to specific tissues in the body, for example, cancer tissue.

Anticancer drug loaded PEGylated zein micelles can be prepared bydissolving the anti cancer agent of interest with the dissolvedPEGylated zein when preparing the micelles. These drug loaded micellescan significantly improve the cellular uptake of anticancer drugs and ismore efficacious than the free drug. The cellular uptake of theanticancer drugs can be determined by measuring the intracellular drugconcentration using HPLC analysis. See FIGS. 30 and 31, and theirdescriptions. The efficacy of the drug loaded micelles compared to thefree drug can be evaluated by measuring the cell viability of drugresistant human cancer cells and determining the concentration requiredto kill 50% of the cells (i.e., determining the IC₅₀ value). The drugloaded micelles had a significantly lower IC₅₀ than the free drug. See,for example, FIGS. 17, 28, and 29, and their descriptions.

It was also surprisingly discovered that anticancer drugs encapsulatedin PEGylated zein micelles are effective against drug resistant cancers.This discovery was found by analysis of drug resistant human cancercells using Calcein acetoxy methyl ester (Calcein AM), a fluorescentmarker that is a substrate for the P-glycoprotein efflux pump.Overexpression of P-gp efflux pump in some cancers leads to drugresistance. The micelles were able to inhibit the P-gp efflux pump andincrease the intracellular concentration of Calcein as measured byspectrofluorimetry. See FIG. 32 and its description. Thus, the PEG-zeinmicelles can inhibit the P-gp efflux pump and enhance the cell uptake ofanti-cancer drugs in drug resistant cancers, such as drug resistantstrains of breast cancer, ovarian cancer, colon cancer, lung cancer andglioblastoma. The encapsulation of hydrophobic compounds in the core ofthe micelles also stabilizes labile compounds against degradation fromenvironmental agents, as determined by measuring the drug concentrationat different time periods (e.g., up to 12 hours) of a free drug solutionor drug loaded micelle dispersion stored at room temperature underlight. The drug concentration was measured by UV-visible spectroscopy.

Additionally, PEGylated zein micelles can be used to develop watersoluble/water dispersible formulations of hydrophobic drugs. Because themicelles are small in size (e.g., about 100 nm to about 300 nm indiameter), they can be used for IV administration of, for example,hydrophobic drugs. They can also be used to improve the bioavailabilityof water insoluble drugs by parenteral, oral, nasal, transdermal, ocularand other routes of drug administration. Lyophilized drug (waterinsoluble drug) loaded micelle can be readily diluted with water beforeinjection. The lyophilized drug loaded micelle can then be incorporatedin a capsule or other suitable formulation matrix. After administrationthe micelles can form in the gastrointestinal intestinal fluids,resulting in enhanced solubility and absorption of water insolubledrugs. Furthermore, the PEGylated-zein micelles are biocompatible andbiodegradable, thereby increasing their safety profile in humans.

In one aspect of the invention, the micelles can be employed astherapeutic and/or diagnostic micelle formulations, e.g., an anticanceragent-containing micelles. Such micelles can provide targeted deliveryand temporal control of the release of an active agent, which is often atherapeutic agent such as a small molecular drug, nucleic acid, protein,vaccine, receptors, hormones, cells, antibody, chemical or other agentor substance. In addition to the therapeutic methods described, theinvention provides means for producing micelles with diagnostic agents,such as dyes, imaging agents, probes, and the like.

Further modifications to the prolamine-polymer conjugates can be madefor specific applications, such as attaching targeting ligands to thehydrophilic shell for targeted delivery to tumors. For example, folicacid, antibodies, and the like can be attached to the PEG shell fortargeting cancer cells that overexpress receptors for specific targetingligands.

Zein micelles formed using the methods described herein may have otheruses, particularly outside of the body. For example, drug-loadedPEGylated zein micelles can be used as a coating material forcardiovascular and other biomedical devices. Although described hereinwith respect to drug delivery, micelles may also be used to encapsulateand sustain the release of molecules of interest to the food, dairy andcosmetic industries. In addition to human drugs, veterinary drugs mayalso be encapsulated in the micelles. PEGylated zein micelles may beused to protect molecules from degradation, such as by hydrolysis,oxidation, photo-degradation, and other degradation reactions. Thisutilization may include molecules of interest to the pharmaceutical,food, dairy, agricultural, nutraceutical and cosmetic industries.

Variations of Formula I:

Formulas I-V can be further modified to provide additional embodiments.In any embodiment that recites zein as an example, another type ofprolamine can be substituted for zein to provide a separate embodiment.For example, in addition to zein (Z) and PEG, other hydrophobic (X) orhydrophilic polymers (Y) can also be conjugated to any of Formulas I-Vto form graft copolymers or ABC type multiblock copolymers, where A, Band C are polymer block moieties of different monomeric units. Examplesof these variations include Formulas VI-IX:

Z-b-PEG-b-X  (VI)

Z-b-PEG-b-Y  (VII)

PEG-b-Z-b-Y  (VIII)

PEG-b-Z-b-X  (IX)

where X is a hydrophobic polymer moiety, Y is a hydrophilic polymermoiety, and Z and PEG are as defined for Formula I.

The hydrophilic polymer PEG of general Formula I can be replaced withother hydrophilic polymers (Y), such as polyvinyl pyrrolidone (PVP),polyvinyl alcohol (PVA), chitosan, polyethyleneimine (PEI), polyacrylicacid (PAA), polysialic acid (PSA), polysaccharides such as dextran, andthe like. Similarly, hydrophobic polymers (X) can be conjugated toprolamines (e.g., zein). Such hydrophobic polymers (X) can include, forexample, polycaprolactone, poly lactic acid-co-glycolic acid,polypropylene oxide, polyaspartate, polygultamate, spermine, polylysine,or polyacrylates such as polymethacrylate, polydimethylamino ethylacrylate, and the like. Fatty acids can also be conjugated to prolaminesto form the hydrophobic core. Examples of such fatty acids include, forexample, stearic acid, palmitic acid, phosphatidylethanolamine, andoleic acid.

Other and/or additional modifications can be made to the prolaminehydrophobic core and/or to the hydrophilic PEG shell. Thesemodifications can include conjugating stimuli responsive elements, suchas polyhydroxyethylmethacrylate, to the core to prepare pH sensitivemicelles, or poly (N-isopropylacrylamide) to prepare thermosensitivemicelles. In addition, the prolamine hydrophobic core or hydrophilicshell can be cross-linked (for example, using cross-linkers such asglutaraldehyde, genipin, or citric acid, and the like) to control drugrelease and to increase drug encapsulation and loading efficiency.

Pharmaceutical Formulations of Micelles:

The micelles described herein can be used to prepare therapeuticpharmaceutical compositions. The micelles may be added to thecompositions in the form of an aqueous dispersion or as a dry powder oflyophilized micelles. The micelles described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The micelles described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration, amicelle dispersion can be enclosed in hard or soft shell gelatincapsules, or lyophilized micelles can be compressed into tablets, orincorporated directly into the food of a patient's/subject's diet.Micelles dispersions or lyophilized micelles may also be combined withone or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations typically contain atleast 0.1 wt % of an active therapeutic or diagnostic agent. The weightpercentage of agent in the compositions and preparations can vary andmay also conveniently be from about 2% to about 60% of the weight of agiven unit dosage form. The amount of active compound in suchtherapeutically useful compositions containing micelles is such that aneffective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethyleneglycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the micelles, in addition to sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing a unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, themicelle dispersion or lyophilized micelles may be incorporated intoadditional sustained-release preparations and devices.

A micelle dispersion may be administered intravenously, subcutaneously,intramuscularly, intratumorally, peritumorally, or by infusion orinjection. Dispersions of the micelles can be prepared in water,optionally mixed with a buffer, or in other pharmaceutically acceptablesolvents, or mixtures thereof. Under ordinary conditions of storage anduse, preparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the micelles adapted for the extemporaneous preparation ofsterile injectable or infusible solutions or dispersions. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers, or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by agentsdelaying absorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating themicelles in the required amount in an appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze drying techniques, which yield a powder of themicelles plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

For topical administration, it will generally be desirable to administerthe micelles to the skin as a composition or formulation, for example,in combination with a dermatologically acceptable carrier, which may bea solid, liquid, gel, cream, ointment, or paste. Useful solid carriersinclude finely divided solids such as talc, clay, microcrystallinecellulose, silica, alumina, and the like. Useful liquid carriers includewater, or water-alcohol/glycol/dimethyl sulfoxide (DMSO) blends, inwhich a micelle can be dispersed at effective levels, optionally withthe aid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. Fluid compositions can be applied from absorbent pads,used to impregnate bandages and other dressings, or sprayed onto theaffected area using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of dermatological compositions for delivering active agents(e.g., agent loaded micelles) to the skin are known to the art; forexample, see U.S. Pat. Nos. 4,608,392 (Jacquet et al.), 4,992,478(Geria), 4,559,157 (Smith et al.), and 4,820,508 (Wortzman), hereinincorporated by reference in their entireties. Such dermatologicalcompositions can be used in combinations with the micelle formulationsdescribed herein.

Useful dosages of drug loaded micelles described herein can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949 (Borch et al.), herein incorporated byreference in its entirety. The amount of a compound, or an active salt,prodrug, or derivative thereof, loaded into a micelle required for usein treatment will vary not only with the particular compound or saltselected but also with the route of administration, the nature of thecondition being treated, and the age and condition of the patient, andwill be ultimately at the discretion of an attendant physician orclinician.

The therapeutic agent loaded micelle can be conveniently administered ina unit dosage form, for example, containing 5 to 1000 mg/m²,conveniently 10 to 750 mg/m², most conveniently, 50 to 500 mg/m² ofactive ingredient per unit dosage form. The desired dose mayconveniently be presented in a single dose or as divided dosesadministered at appropriate intervals, for example, as two, three, fouror more sub-doses per day. The sub-dose itself may be further divided,e.g., into a number of discrete loosely spaced administrations.

The drug loaded micelles described herein can be effective anti-tumoragents and have higher potency and/or reduced toxicity as compared tonon-micelle encapsulated anti-tumor agents. The invention providestherapeutic methods of treating cancer in a mammal, which involveadministering to a mammal having cancer an effective amount of acomposition described herein. A mammal includes a primate, human,rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovineand the like. Cancer refers to any various type of malignant neoplasm,for example, colon cancer, breast cancer, melanoma and leukemia, and ingeneral is characterized by an undesirable cellular proliferation, e.g.,unregulated growth, lack of differentiation, local tissue invasion, andmetastasis.

The ability of a compound of the invention to treat cancer may bedetermined by using assays well known to the art. For example, thedesign of treatment protocols, toxicity evaluation, data analysis,quantification of tumor cell kill, and the biological significance ofthe use of transplantable tumor screens are known.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1 Preparation of PEGylated Zein and Formation ofMicelles

PEGylated zein nanomicelles having a size range distribution of betweenapproximately 80 nm and approximately 200 nm were prepared as describedherein. FIGS. 2 and 3 illustrate the stepwise preparation of PEG-Zeinaccording to various embodiments. PEGylated zein was prepared by adding0.1 g of methoxy PEG-succinimidyl succinate (m.w. 1000, 2000, or 5000Da) to 0.1 g of white zein in 5 mL of 90% ethanol. The mixture inspecific ratio (1:1, w/w) was incubated for three hours (stirred at 50rpm) at 37° C. After 3 hours, 1 mL of aqueous glycine (1 M) solution wasadded to quench any excess PEG ester. Five mL of citrate buffer, pH 7.4,was then added to precipitate the PEGylated zein. The precipitateddispersion of PEGylated zein was then directly dialyzed (m.w. cutoff=˜10,000 Da) against deionized water in a magnetic stirrer (100 rpm)at room temperature (−23° C.) for 24 hours to remove free PEG, glycine,and ethanol. The resulting product was frozen to −80° C. followed byfreeze drying at −47° C. at 60 mTorr vacuum for 12 to 14 hours.

The m-PEG-N-hydroxy succinimidyl ester (5 kDa) was used to form an amidebond with the amino group in zein. The conjugate was confirmed usingFTIR. Amide I and II protein peaks of zein are observed in 1650 and1500-1540 cm⁻¹, respectively. The NHS ester peak for PEG is observed in1740 cm⁻¹ which disappeared after conjugation with zein (FIG. 4).Further, the conjugate was characterized by size exclusion chromatograph(SEC). As can be seen in FIG. 4, PEG-zein conjugate eluted at 7 minutesand zein eluted at 23 minutes. On the other hand, PEG eluted at 29minutes.

The efficiency of PEGylation observed over various molecular weight PEGconjugated zein is shown in Table 1 below, where the efficiencypercentages were determined using a trinitrobenzene sulfonic acid (TNBS)assay. The surface amino groups in zein were found to be involved inPEGylation. The TNBS assay was used to estimate the free amino groups inzein before and after PEGylation. A standard curve was generated withincreasing concentration of pure zein and PEGylated zein versusabsorbance at 440 nm wavelength. PEGylation efficiency was calculatedusing the formula:

% of PEGylation efficiency=[a−b/a]×100

where a=slope of the concentration of non-PEGylated zein versusabsorbance, and b=slope of the concentration of PEGylated zein versusabsorbance. The concentration range of zein used for constructing thestandard curve was 0.357 mg/mL to 12 mg/mL, and correlation coefficientwas 0.9994.

TABLE 1 Zein PEGylation Efficiency. Sample PEG molecular weight (Da)PEGylation Efficiency (%) 1 1000 74 ± 7 2 2000 60 ± 4 3 5000 52 ± 6Results are representative of triplicate samples (average ± SD).

Smaller sized PEG-Zein micelles were formed using PEG>3000 Da, asillustrated by the data shown in Table 2. The PEGylated zeinself-assembles in aqueous environment to form nanomicelles (˜100 nm)with a hydrophobic core and a hydrophilic shell, as schematicallyillustrated in FIG. 1.

TABLE 2 PEG Molecular Weight Required for Micelle Formation. PEGmolecular weight Particle size Sample (Da) (nm) PDI 1 1000 970 ± 1250.69 ± 0.12 2 2000 902 ± 107 0.65 ± 0.08 3 5000  95 ± 1.7 0.21 ± 0.02Results are representative of triplicate samples (average ± SD).

Core-Shell Structure of PEG-Zein: In dimethyl sulfoxide (DMSO), ¹H NMRresonance peaks corresponding to hydrophobic and hydrophilic portions ofboth zein and PEG 5000 Da and are clearly observed in the NMR spectra(FIG. 5): 3.56 ppm for the PEG methylene resonances and 3.36 ppm for theprotein/amide resonances. In contrast, only PEG resonance peaks weredetected in D₂O and zein peaks were not observed. This result confirmsthe core-shell structure of PEG-Zein micelles. In deuterated water(D₂O), the protein peaks for zein are not observed because the zein isinsoluble in water. However, the PEG peak is observed in D₂O because thePEG is water soluble. For the PEG-zein micelle, the shell consisting ofPEG blocks is well solvated in D₂O and therefore shows clear NMRspectral peaks, while the resonance peaks of zein, which constitutes thecore of the micelles, were not observed due to the lack of the solventand solvation within the micelle core. DMSO, however, solubilizes andbreaks down micelles and thus is able to solvate both PEG and protein,allowing for the peaks corresponding to both portions of the moleculesto be recorded (FIG. 5).

The concentration required for formation of micelles is known as thecritical micellar concentration (CMC). The CMC value determines thestability of a micelle upon dilution with water. The CMC for PEGylatedzein is 0.025±0.0095 g/L, which was determined using pyrene as a probe(FIG. 7). Because the zein molecular weights are relatively high, theyform more stable micelles than other polymeric micelles (see, forexample, FIG. 6), as indicated by the lower CMC value of zein micelles.FIG. 6 illustrates the plot of the ratio of absorbance of pyrene (0.6ILIM) at the excitation wavelengths of 339 nm and 334 nm (emissionwavelength is 390 nm) against logarithmic concentration (g/L) ofPEGylated zein. As the concentration of PEGylated zein is increased,there is a significant shift in the intensity of absorbance of pyrene atthe CMC (i.e., concentration at which micelles are formed).

The particle size of micelles did not change significantly on dilutionwith buffer indicating the stability of micelles (FIG. 6). The preparedPEG-zein micelles were non immunogenic as determined by the absence ofany zein specific antibodies after subcutaneous administration in mice(FIG. 8). A summary of particle sized and encapsulation efficiencies forvarious hydrophobic compounds loaded into PEG-zein micelles is shown inTable 3 below.

TABLE 3 PEG-Zein Micelle Encapsulation Data of Various Compounds. M.W.Particle size Encapsulation Sample Compound Log P (Da) (nm) PDIEfficiency(%) 1 Curcumin 2.5 368.3  124 ± 4.1 0.25 ± 0.03 95 ± 4 2Doxorubicin 1.20 543.5 153 ± 3 0.18 ± 0.06 92 ± 6 3 Nile red 5 318.3 165± 7 0.21 ± 0.08  77 ± 11 Results are representative of triplicatesamples (average ± SD); PDI = polydispersity index.

Example 2 PEGylated Zein Micelles Encapsulating Doxorubicin

Doxorubicin is a widely used anticancer drugs for the treatment ofbreast cancer and ovarian cancer, among others. However, the clinicaluse of doxorubicin is limited by serious side effects, such asmyelosupression and chronic cardio toxicity, which can lead tocongestive heart failure (Hortobagyi (1997), Drugs 54 Suppl 4:1-7).Another limitation of doxorubicin is the development of resistance tochemotherapy (Gottesman et al. (2002), Nat Rev Cancer 2:48-58).Doxorubicin has a molecular weight (m.w.) of 543.5, a Log P of 1.2, ispractically insoluble in water, and is soluble in methanol, ethanol andDMSO.

Compared to low molecular weight surfactant micelles, polymer micellesare generally more stable with a low critical micelle concentration(CMC) and slower dissociation, allowing retention of loaded doxorubicinfor longer period of time, and eventually, achieving a higheraccumulation of the drug at the target site. Such selective passivetargeting capability is due to the enhanced permeability and retentioneffect, resulting from a leaky vasculature and a lack of lymphaticdrainage in tumor tissues (Maeda et al. (2000), J Control Release65:271-284).

Water insoluble doxorubicin base was extracted from its hydrochloridesalt. Doxorubicin hydrochloride (0.012 g) was dissolved in 100 mL ofdeionized water (0.22 μm filtered), and was stirred magnetically for 10minutes to allow complete solubilization of doxorubicin. The pH of 10the solution was 7.2. Triethylamine (0.2 mL) was added followed bymagnetic stirring for 30 minutes to allow uniform mixing. The pH of theresulting solution was 12. To this aqueous solution 100 mL of chloroformwas added and was stirred magnetically for 15 minutes. The resultingemulsion was shaken vigorously in a separating funnel, and thechloroform layer was recovered. The procedure was repeated three timesto recover the base completely. Fractions were combined and concentratedto dryness under reduced pressure (on a rotary evaporator). The dryresidue was redissolved in chloroform and was rinsed with a saturatedaqueous solution of sodium chloride. The chloroform layer was separatedinto a round bottom flask and was completely concentrated to dryness ona rotary evaporator. The doxorubicin base in a round bottom flask waskept in an oven (under dark conditions) at 37° C. for 48 hours to allowcomplete drying. The product was stored at 4° C. until used.

FIGS. 20 and 21 illustrate the stepwise preparation ofdoxorubicin-loaded PEG-Zein micelles using film and dialysis methods,respectively. In both film and dialysis methods, 0.1 g of PEG-Zein and0.001 g of doxorubicin were dissolved in 20 mL of 90% ethanol. Themixture was incubated overnight (magnetic stir bar, stirred at 50 rpm)at 37° C. to allow partitioning of doxorubicin into the hydrophobic zeincore. After overnight incubation, the hydroalcoholic solvent wascompletely removed under reduced pressure by rotary evaporation to forma thin film. The dried film of doxorubicin-loaded PEG-Zein micelles wasreconstituted in a citrate buffer pH 7.4 and sonicated for 5 minutes toform a uniform suspension. The mixture was then dialyzed (m.w. cutoff=˜10,000 Da) against water in a magnetic stirrer.

For the dialysis method, after overnight incubation, the mixture wasdialyzed (m.w. cut off=˜10,000 Da) against water in a magnetic stirrer(100 rpm) at room temperature for 24 hours to remove any residualmaterial. The resulting product was then frozen to −80° C. followed byfreeze drying at −47° C. at 60 mTorr vacuum for 12 to 14 hours (FIG.21). The lyophilized product was stored in a dessicator underrefrigerated condition at 4° C. Table 4, below, illustrates variouscharacteristics of doxorubicin-loaded PEGylated zein micelles preparedusing a film method and a dialysis method, respectively.

TABLE 4 Doxorubicin Particle size Encapsulation Sample (% w/w) (nm) PDIEfficiency (%) Film Method 1 0.025 170 ± 10 0.47 ± 0.1  72 ± 2.8 2 0.1327 ± 19 0.29 ± 0.02 26 ± 4.7 3 0.2 427 ± 43 0.27 ± 0.06 12 ± 1.3Dialysis Method 4 0.01 145 ± 2  0.16 ± 0.01 92 ± 6   5 0.025 153 ± 3 0.18 ± 0.06 89 ± 3.5 6 0.05 185 ± 10  0.5 ± 0.12 59 ± 8.3 Results arerepresentative of triplicate samples (average ± SD); PDI =polydispersity index.

The amount of free doxorubicin, encapsulated doxorubicin, amountreleased during in vitro release study, and cell uptake was quantifiedusing a gradient HPLC with the mobile phase consisting oftrifluoroacetic acid (0.1% v/v) and acetonitrile 5% v/v ˜3 min, 80% v/v˜11 min and 5% v/v ˜22 minutes, at a flow rate of 1 mL/min usingfluorescence detector (505 nm as the excitation and 550 nm as theemission wavelengths).

${{Encapsulation}\mspace{14mu} {efficiency}\mspace{11mu} (\%)} = {\frac{\begin{matrix}{{Actual}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {doxorubicin}} \\{{loaded}\mspace{14mu} ( {{mg}\text{/}{mg}} )\mspace{14mu} {into}\mspace{14mu} {PEG}\text{-}{Zein}}\end{matrix}}{\begin{matrix}{{Amount}\mspace{14mu} {of}\mspace{14mu} {doxorubicin}\mspace{14mu} {added}} \\{( {{mg}\text{/}{mg}} )\mspace{14mu} {to}\mspace{14mu} {PEG}\text{-}{Zein}\mspace{14mu} ({theoretical})}\end{matrix}} \times 100}$

FIGS. 22 and 23 show transmission electron microscopic (TEM) and atomicforce microscopy (AFM) images, respectively, of doxorubicin-loadedPEG-Zein micelles.

Doxorubicin is practically insoluble in water (15 ng/mL). However whenincorporated into PEGylated zein micelles, the solubility increased byapproximately 1000 fold (10 ng/mL). FIG. 24 illustrates the UV-Visiblespectra of doxorubicin in phosphate buffer pH 7.4, 90% ethanol, anddoxorubicin-loaded PEGylated zein micelles in PBS pH 7.4, respectively.The absorbance of the doxorubicin-loaded PEG-Zein is higher than theabsorbance of doxorubicin solubilized in 90% (v/v) ethanol, which showsthe enhanced aqueous solubility of doxorubicin-loaded PEG-zein micelles(1000 fold increase).

FIG. 25 shows fluorescence spectra of doxorubicin in phosphate buffer pH7.4, 90% ethanol, and doxorubicin-loaded PEGylated zein micelles in PBS7.4, respectively. There is a significant increase (approximately 50fold) in doxorubicin fluorescence in PBS pH 7.4 after entrapment inPEGylated zein micelles due to the enhanced solubility of doxorubicin.Differential scanning calorimetry (DSC) thermograms ofdoxorubicin-loaded PEG-Zein micelles are shown in FIG. 26. The absenceof a melting peak of doxorubicin indicates encapsulation of doxorubicininside the core of the micelles. In vitro release of doxorubicin fromPEG-Zein micelles is illustrated in FIG. 27. Release of doxorubicin wassustained for about 24 hours. PEGylated zein micelles are thus apromising carrier for doxorubicin.

The therapeutic activity of doxorubicin-loaded PEGylated zein micellesprepared as described herein was tested in vitro against doxorubicinsensitive human breast cancer cells (MCF-7) and doxorubicin resistanthuman ovarian cancer cells (NCl/ADR/RES), and a doxorubicin sensitivehuman breast cancer cell line (MCF-7). FIG. 28 illustrates an in vitrocytotoxicity profile of doxorubicin (dissolved in 90% ethanol) andPEG-Zein micelles in MCF-7 cells. Cells at a seeding density of 2000 perwell were exposed to a doxorubicin solution and doxorubicin micelles atconcentration of 7.8 nM to 500 nM. After 24 hours, the respective drugtreatments were removed. The cells were washed twice with ice coldphosphate buffer and replaced with fresh media. Media was replaced forevery 48 hours. At day 5, cytotoxicity analysis was performed using theMTT assay.

The IC₅₀ value for doxorubicin micelles was half that of the puredoxorubicin treatment. FIG. 29 illustrates an in vitro cytotoxicityprofile of doxorubicin base (dissolved in 90% v/v ethanol) and PEG-Zeinmicelles in an NCl/ADR-RES cell line. Cells at seeding density of 2000cells per well were exposed to doxorubicin base and doxorubicin micellesat concentration of 31.25 nM to 1000 nM. After 24 hours the respectivedrug treatments were removed. The cells were washed twice with ice coldphosphate buffer and replaced with fresh media. Media was replaced forevery 48 hours. On day 5 cytotoxicity analysis was performed using anMTT assay. The IC₅₀ value for the doxorubicin micelle was 4 times lowerthan that of the free doxorubicin treatment. The results of the in vitrocytotoxicity assay of doxorubicin-loaded PEGylated zein micelles inhuman cancer cell lines showed that the doxorubicin-loaded in PEGylatedzein micelles had a significantly higher effective potency than the freedoxorubicin solution. The difference in potency can be attributed to thedifference in the cell uptake kinetics of the free drug compared to thedoxorubicin loaded PEGylated zein micelles.

Free doxorubicin is taken up by passive diffusion dictated by theconcentration gradient, while the doxorubicin-loaded micelles are takenup an active endocytosis process. FIG. 30 illustrates the influence oftemperature on cellular uptake of doxorubicin loaded PEG-zein micellesin NCl/ADR-RES cell line. Cells were pre-incubated at 4° C. for 2 hours.After 2 hours the cells were washed twice with PBS pH 7.4 and weretreated with the micelles (amounts corresponding to 5 μg/mL ofdoxorubicin). After two hours the treatments were removed, the cellswere washed twice with ice cold PBS pH 7.4, and the amount ofdoxorubicin content in the cell lysate at different time intervals wasestimated using HPLC analysis.

The cell uptake was significantly reduced at low temperature signifyingthat the cell uptake of PEG-zein micelles is an active endocytosisprocess. FIG. 31 illustrates the kinetics of cellular uptake ofdoxorubicin-loaded PEG-Zein micelles and a solution in NCl/ADR-RES cellline (5 μg/mL of doxorubicin or doxorubicin-loaded PEG-zein micelles(5000 cells/well)). Higher cell uptake of doxorubicin micelles wasobserved in comparison to plain doxorubicin solution at all time points.Furthermore, the endocytotic uptake of doxorubicin micelles overcame thedrug efflux pumps in resistant cancer cells, thus increasing the drugefficacy.

FIG. 32 illustrates the influence of PLURONIC F68 treatment (1 mg/mL),and blank PEG zein micelles (0.050 mg/mL) on P-gp activity (Calcein AMassay) in NCl/ADR-RES cells. Calcein AM is non-fluorescent and readilydiffuses into cells. Calcein AM, but not calcein, is a substrate forP-gp. In the presence of P-gp inhibitors, calcein AM enters the cell andis converted to calcein by intracellular esterases. Fluorescenceincreased with increased intracellular calcein concentrations. From thedata illustrated in FIG. 32, it is evident that significant P-gpinhibition is observed with blank PEG-zein micelles. Targeting ligandscan also be attached to facilitate delivery of the drug loaded PEG-zeinnanomicelles to a target site in vivo.

FIG. 33 shows the in vivo biodistribution of doxorubicin loaded PEG-zeinmicelles in an allograft mouse tumor model. Female nude mice (CharlesRiver Laboratories, Wilmington, Mass.) were used in the study. JC mousebreast cancer cells (1×10⁷ cells) were suspended in PBS and injectedsubcutaneously. When the tumor volume reached ˜150 to 200 mm³, animalsreceived intravenous injections of doxorubicin solution or doxorubicinloaded PEG-zein micelles (3 mg/kg). After 3 hours, the mice weresacrificed and organs (liver, heart, lungs, spleen, brain and tumor)were collected and homogenized in 2 mL deionized water using a tissuehomogenizer. After addition of 100 ng of daunorubicin (internalstandard), tissue homogenates were lyophilized.

Dry tissues were weighed and extracted with 5 mL of methanol/chloroformmixture (65:35) using a shaker for 5 hours in the dark at roomtemperature. The extract was centrifuged at 13,000 rpm for 10 minutes at4° C. The supernatant was evaporated under nitrogen gas andreconstituted in methanol/acetonitrile (50:50). The amount ofdoxorubicin in the organs was quantified using a reverse phase HPLCmethod (27 (acetonitrile): 73 (20 mM potassium hydrogen phosphate(monobasic) buffer (pH: 2.5)) using a fluorescence detector (excitationwavelength 505 nm and emission wavelength 550 nm). The amount ofdoxorubicin in the organs was expressed as the amount (ng) per mg of dryorgan.

As can be observed in FIG. 30, a higher drug accumulation was found intumors while there was no drug accumulation of the free doxorubicinsolution in the tumors. The drug concentration in heart and kidneytissue was significantly lower with doxorubicin PEG-zein micellescompared to the free doxorubicin solution samples. Doxorubicinchemotherapy is limited by cardiac and renal toxicity. These resultsdemonstrate enhanced tumor accumulation and reduced toxicity ofdoxorubicin loaded PEG-zein micelles.

The efficacy of doxorubicin PEG zein micelles was studied by measuringthe change in tumor volume in an allograft breast tumor mouse model.Female BALB/c mice (Charles River Laboratories, Wilmington Mass.) wereused in the study. JC mouse breast tumor cells (1×10⁷ cells) weresuspended in PBS and injected subcutaneously. When the tumor volumereached ˜150 to 200 mm³, animals received two doses of intravenousdoxorubicin solution or doxorubicin loaded PEG-zein micelles (3 mg/kg,on days 0 and 7). The tumor volume was measured using a Vernier Caliper.

FIG. 34 shows the increase in tumor volume after different treatments.As can be seen in FIG. 34, the increase in tumor volume wassignificantly lower with the doxorubicin PEG-zein micelles. The micetreated with doxorubicin PEG-zein micelles also lived longer than theother treatment groups (FIG. 35). The results demonstrate the enhancedefficacy of doxorubicin loaded PEG-zein micelles.

Example 3 PEGylated Zein Micelles Encapsulating Curcumin

Curcumin is the principal curcuminoid of the Indian spice turmeric,which is a member of the ginger family (Zingiberaceae). Two othercurcuminoids are desmethoxycurcumin and bis-desmethoxycurcumin. Curcumincan exist in at least two tautomeric forms, of which the enol form ismore energetically stable in the solid phase and in solution. Curcuminhas a molecular weight (m.w.) of 368.4, a Log P of 2.5, is practicallyinsoluble in water and is soluble in methanol.

Clinical trials are studying the effect of curcumin on various diseasesincluding multiple myeloma, pancreatic cancer, myelodysplasticsyndromes, colon cancer, psoriasis, and Alzheimer's disease. In vitroand animal studies indicate that curcumin has antitumor, antioxidant,antiarthritic, anti-amyloid, anti-ischemic, and anti-inflammatoryproperties, as well as other biological activities (Aggarwal et al., AdvExp Med Biol 2007, 595:1-75). FIGS. 9 and 10 illustrate the stepwisepreparation of curcumin-loaded PEG-Zein micelles using film hydrationand dialysis methods, respectively. In both film hydration and dialysismethods, 0.1 g of PEG-Zein and 0.002 g of curcumin was dissolved in 20mL of 90% ethanol. The mixture was incubated overnight (stirred at 50rpm) at 37° C. to allow partitioning of curcumin into the hydrophobiczein core. The hydroalcoholic solvent was then completely removed usinga rotary evaporation device to form a film. The dried film ofcurcumin-loaded PEG-Zein micelles was reconstituted in citrate buffer,pH 7.4, and was sonicated for 5 minutes to form a uniform suspension.The mixture was then dialyzed (m.w. cut off=˜10,000 Da) against waterwith stirring (100 rpm) at room temperature for 24 hours to remove freecurcumin.

In the dialysis method, after overnight incubation, the mixture wasdialyzed (m.w. cut off 10,000 Da) against water in a magnetic stirrer(at 100 rpm) at room temperature for 24 hrs to remove free curcumin. Theresulting product was then frozen to −80° C. followed by freeze dryingat −47° C. at 60 mTorr vacuum for 12 to 14 hours. The lyophilizedproduct was stored in dessicator at 4° C.

Tables 5 and 6 below illustrate various characteristics ofcurcumin-loaded PEGylated zein micelles prepared using a thin filmmethod and a dialysis method, respectively.

TABLE 5 Curcumin Particle size Encapsulation Sample (% w/w) (nm) PDIEfficiency (%) 1 0.25 166 ± 10 0.4 ± 0.1  92 ± 3.5 2 0.5 139 ± 2  0.43 ±0.05 76 ± 11 3 1 176 ± 9   0.4 ± 0.12 47 ± 17 4 2 185 ± 13 0.52 ± 0.0638 ± 4  Results are representative of triplicate samples (average ± SD);PDI = polydispersity index.

TABLE 6 Curcumin Particle size Encapsulation Sample (% w/w) (nm) PDIEfficiency (%) 1 1 124 ± 4.1 0.25 ± 0.03 95 ± 4  2 1.25 127 ± 2.6 0.31 ±0.01 94 ± 7  3 1.66 148 ± 7   0.34 ± 0.09 87 ± 15 4 2.5 152 ± 2.5 0.35 ±0.03 74 ± 09 5 5 154 ± 1   0.45 ± 0.04 60 ± 13 6 4 175 ± 1.7 0.38 ± 0.0363 ± 11 Results are representative of triplicate samples (average ± SD);PDI = polydispersity index.

The concentration of free curcumin and encapsulated curcumin was assayedby RP-HPLC using a C18 column. The mobile phase consisted of 60%acetonitrile and 40% citric buffer (1% (w/v) citric acid solutionadjusted to pH 3.0 using 50% (w/w) sodium hydroxide solution). The flowrate was 1.0 mL/min and the detection wavelength was 420 nm.

${{Encapsulation}\mspace{14mu} {efficiency}\mspace{11mu} (\%)} = {\frac{\begin{matrix}{{Actual}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {curcumin}} \\{{loaded}\mspace{14mu} ( {{mg}\text{/}{mg}} )\mspace{14mu} {into}\mspace{14mu} {PEG}\text{-}{Zein}}\end{matrix}}{\begin{matrix}{{Amount}\mspace{14mu} {of}\mspace{14mu} {curcumin}\mspace{14mu} {added}} \\{( {{mg}\text{/}{mg}} )\mspace{14mu} {to}\mspace{14mu} {PEG}\text{-}{Zein}\mspace{14mu} ({theoretical})}\end{matrix}} \times 100}$

FIGS. 11 and 12 show transmission electron microscopic (TEM) and atomicforce microscopy (AFM) image of curcumin loaded PEG-Zein micelles.Curcumin is practically insoluble in water (11 ng/mL) (B. Aggarwal etal. (2007), Adv Exp Med Biol 595:1-75). However when incorporated intoPEGylated zein micelle, the solubility increased by approximately 2000fold (20 μg/mL). FIG. 13 is a UV-Visible spectra of curcumin in 10%methanol and curcumin-loaded PEGylated zein micelles in PBS pH 7.4. Itis evident from the spectra that the absorbance of the curcumin loadedPEG-Zein is similar to the absorbance of curcumin solubilized in 10%(v/v) methanol. Thus PEG-Zein significantly enhanced the aqueoussolubility of curcumin by approximately 2000 fold. FIG. 14 is afluorescence spectra of curcumin in 10% methanol and curcumin loadedPEGylated zein micelles in PBS pH 7.4. The shift of the Xmax of theemission spectra of curcumin from 540 nm to 525 nm shows that thecurcumin is entrapped in the micelles. Furthermore, there is asignificant increase (approximately 4 fold) in curcumin fluorescence inwater after entrapment in PEGylated zein micelles due to enhancedaqueous solubility of curcumin. The encapsulation in the core stabilizedcurcumin against degradation from environmental agents, such ashydrolysis and photodegradation.

Table 7, below, illustrates the stability of curcumin-loaded PEGylatedzein micelles in the presence of light and pH variation. Stability ofcurcumin is improved when encapsulated in PEG-zein micelles (half life31.8 minutes), in comparison to plain solution (half life 4 minutes)(phosphate buffer pH 7.4). Stability was remarkably enhanced inphosphate buffer pH 5 (half life=6.9 minutes, compared to micelles,t_(1/2)=366 minutes).

TABLE 7 Sample Formulation Conditions t_(1/2) (min) 1 Curcumin* pH 7.4 42 Curcumin loaded PEG-zein micelles pH 7.4 31.8 3 Curcumin* pH 5   6.9 4Curcumin loaded PEG-zein micelles pH 5   366 *10% v/v methanol was usedto solubilize curcumin.

Differential scanning calorimetry (DSC) thermograms of curcumin-loadedPEG-Zein micelles are shown in FIG. 15. The absence of a melting peak ofcurcumin indicates encapsulation of curcumin inside the core ofmicelles. In vitro release of curcumin from PEG-Zein micelles ispresented in FIG. 16. Release of curcumin was sustained for about 24hours. Curcumin is known to have anti-cancer and anti-inflammatoryactivities, however the delivery of curcumin is limited by its poorwater solubility. PEGylated zein micelles can be a suitable carrier forcurcumin. FIG. 17 illustrates the in vitro cytotoxicity of curcumin(dissolved in 10% DMSO) and curcumin micelles (in PBS 7.4) in drugresistant human ovarian cancer cells (NCl/ADR-RES cells). The cell (2000cells/well) were treated with 7.8 nM to 500 nM of curcumin for 4 days.On the fifth day cytotoxicity analysis was performed using the MTTassay. The curcumin-loaded micelles were more potent (3 fold more) thanthe pure curcumin.

In Vitro Skin Penetration of Free Curcumin and Encapsulated Curcumin.

As can be seen in FIG. 18, the skin penetration of curcumin was enhancedby 5-20 fold. Unlike the free curcumin, the skin penetration of curcuminmicelles increased with treatment time. A significant amount of theapplied (5-20%) dose penetrated the skin and curcumin was even found tocross and reach the receptor phase with longer treatment time.

As can be seen in FIG. 19, the curcumin micelles were mainly localizedto the hair follicles (c). This is also evident from “b” where thefluorescence is observed in streaks from the surface to 100 μm deepinside the skin. The localization of PEG-zein micelles is particularlyuseful for treating follicular diseases such as acne, hair loss,seborrhetic eczema, follicullitis and some skin cancers. FIG. 19 (d)shows the fluorescence pixels in the stratum corneum (0-15 μm) andviable epidermis (20-100 μm). The encapsulation of curcumin in themicelles significantly increased the skin penetration of curcumin, Eachvalue is avg.±SD (n=4). Excised porcine skin was sandwiched between thetwo compartments of a vertical diffusion cell. The receptor mediumconsisted of phosphate buffer (pH 7.4 with 20% ethanol) maintained at37° C. and stirred using a magnetic bead. Free or encapsulated curcuminwas applied on the skin for 6 hours. At the end of the study, the skinwas washed and observed under confocal fluorescence microscope. Thefluorescence pixels were quantified using IMAGEJ software.

Example 4 PEGylated Zein Micelles Encapsulating Nile Red

Nile red was used as a model hydrophobic dye (Sheihet et al. (2008),Int. J. Pharm. 350: 312-319) to study the application of PEG-zeinnanomicelles as a skin delivery vehicle.

Nile red has a molecular weight of 318.4, a Log P: 5, and a meltingpoint of 203-205° C. It is practically insoluble in water, but issoluble in methanol, ethanol, and DMSO.

FIG. 36 illustrates the step wise preparation of Nile red-loadedPEG-Zein micelles using a dialysis method, according to one embodiment.PEG-Zein (0.1 g) and 0.5 mg of Nile red were dissolved in 20 mL of 90%ethanol. The mixture was incubated overnight (stirred at 50 rpm) at 37°C. to allow partitioning of the Nile red into the hydrophobic zein core.The mixture was then dialyzed (m.w. cut off 10,000 Da) against water(stirred at 100 rpm) at room temperature for 24 hours to remove anyresidual material. The resulting product was then frozen to −80° C.followed by freeze drying at −47° C. at 60 mTorr vacuum for 12 to 14hours. The lyophilized product was stored in a dessicator at 4° C.Characteristics of the Nile red-loaded PEG-Zein micelles are shown belowin Table 9.

TABLE 9 Model Particle size Encapsulation compound (nm) PDI Efficiency(%) Nile red 165 ± 7 0.21 ± 0.08 77 ± 11 Results are representative oftriplicate samples (average ± SD); PDI = polydispersity index.

Nile red is a model lipophilic dye used to study skin penetration. Theprepared Nile red loaded PEG-zein micelles had an average particle sizeof 165 nm with a low polydispersity index (0.21). The PEG zein micellesprovided good encapsulation efficiency (77%). FIG. 37 illustrates theability of the PEG-zein micelles to increase the skin penetration andskin retention of hydrophobic compounds. The data also demonstrates theapplication of PEG-zein micelles as a skin delivery vehicle fortherapeutic and cosmetic agents.

The quantity of Nile red found in the epidermis and stratum corneum(n=3) was studied using dermatomed porcine skin. Porcine ears wereobtained from the slaughter house in the Department of Animal and RangeSciences at South Dakota State University. The ears were collectedimmediately after slaughtering and were washed under tap water. Hair onthe dorsal side was removed with a hair clipper. The skin was excisedfrom the underlying cartilage using a scalpel and forceps. Fat adheringto the dermis side was carefully removed using a blunt scalpel and theskin was observed for any visible damage.

Skin was dermatomed to a thickness of 300 μm using a model B electricdermatome (PADGETT™ Instruments, St. Louis, Mo.). Dermatomed porcineskin was sandwiched between the donor and receptor chambers in a Franzdiffusion cell (PERMEGEAR™, Hellertown, Pa.). The receptor chamber wasfilled with 6 mL of phosphate buffer (PB, pH 7.4) and was stirred usinga magnetic stirbar. The receptor medium was maintained at 37° C. Thedonor chamber was loaded with 100 μL of Nile red (250 ng) in 5% v/vTWEEN-80 solution and Nile red micelles in water (equivalent to 250 ngof Nile red). After 6 hours, the skin samples were washed with PBS andmounted on a microscope slide for analysis by confocal laser scanningmicroscopy (CLSM).

The skin with the stratum corneum (SC) side up was examined using CLSM(FLUOVIEW FV300™, Olympus ix70, Olympus, Center Valley, Pa.). Nile redwas excited using an Argon laser at an excitation wavelength of 488 nm.The images were observed using a PLAN-NEOFLUAR 40/0.85 objective. Thexyz confocal images of the skin were scanned from surface (z=0 μm) to100 μm at a step size of 5 μm/scan. All images were obtained with thesame optical aperture, lens and scan speed.

Blank skin did not show any auto-fluorescence. Each representative imagewas selected from three to four skin samples and in each skin three tofour different regions were scanned. Optical sections (xyz) wereanalyzed using FLUOVIEW™ software (Olympus, Center Valley, Pa.). Thefluorescence intensity distribution in the confocal images wasquantified by integrating the total pixels. At least three to fourregions were analyzed for each skin. The pixels in the SC (0-15 μm) andviable epidermis (VE, 20-100 μm) were calculated separately. Treatmentof skin with Nile red micelles showed significant increase in the skinpenetration into both stratum corneum and viable epidermis compared tofree Nile red solution (FIG. 37). This data shows that PEG-zein micellescan be used to deliver therapeutic or cosmetic agents to stratum corneumor viable epidermis to effectively treat various skin conditions.

Example 5 Additional Conjugate and Micelles Embodiments

Variations of the PEGylated zein micelles described herein can also beprepared. For example, in place of zein, other hydrophobic prolamineproteins, such as gliadin, hordein and kafirin may be used as thePEGylated proteins for micelle formation. Accordingly, PEGylated gliadinmicelles, PEGylated hordein micelles, and PEGylated kafirin micelles canbe prepared and used similar to the PEGylated zein micelles describedherein.

Additionally, other amphiphilic protein conjugates can be prepared byreplacing the PEG moiety of the PEGylated prolamine copolymer withanother water soluble polymer, such as polyvinylpyrrolidone (PVP),polyglycolic acid (PGA), polyvinyl alcohol (PVA), chitosan, polysialicacid (PSA), polyethyleneimine (PEI), polyacrylic acid (PAA),polysaccharides such as dextran, and the like. These water solublepolymers can be conjugated to any of the hydrophobic prolamine proteins,such as zein, gliadin, hordein and kafirin, to form amphiphilic proteinconjugates that self-assemble into micelles. When the micelles areformed in the presence of a dissolved therapeutic agent, drug loadedmicelles can be prepared from these various amphiphilic proteinconjugates and can be used as described for the PEGylated zein micelles.

Similarly, hydrophobic polymers can be conjugated to a prolamine Suchpolymers can include, for example, polycaprolactone, poly lacticacid-co-glycolic acid, polypropylene oxide, polyaspartate,polygultamate, spermine, polylysine, or polyacrylates (for example,polymethacrylate, polydimethylamino ethyl acrylate, and the like). Fattyacids can also be conjugated to a prolamine to form a hydrophobic core.Examples of such fatty acids can include stearic acid, palmitic acid,phosphatidylethanolamine, or oleic acid. These polymers and/or fattyacids can be conjugated to any of the hydrophobic prolamine proteins,such as zein, gliadin, hordein and kafirin, to form protein conjugatesthat self-assemble into micelles. When the micelles are formed in thepresence of a dissolved therapeutic agent, drug loaded micelles can beprepared from these various protein conjugates and can be used asdescribed for the PEGylated zein micelles.

Other or further modifications can be made to the prolamine hydrophobiccore or to the hydrophilic shell, such as a PEG shell. These may includeconjugating stimuli responsive elements, such aspolyhydroxyethylmethacrylate, to the core to prepare pH sensitivemicelles or poly (N-isopropylacrylamide) to prepare thermosensitivemicelles. In addition, the prolamine hydrophobic core or hydrophilicshell can be cross-linked, for example, using cross-linkers such asglutaraldehyde, genipin, citric acid, and the like, to control drugrelease and increase drug encapsulation yield and efficiency.

Example 6 Prolamine Micelles for Topical Delivery of Retinoids

Novel nanocarriers for topical delivery of retinol through skin fortreating various dermatological conditions have been developed. Retinol(Vitamin A) and its derivatives (retinoids) are involved in variousbiological functions in the body including epidermal cell growth anddifferentiation, vision, immumomodulatory and anti-inflammatory effects(Summer, J Nutr 138:1835-1839, 2008). In particular, retinol and itsderivatives are widely used for treating various dermatologicalconditions including acne, psoriasis, keratinization disorders, skindiscoloration, and cutaneous malignancies (skin cancer and melanoma), aswell as for wound healing and photoaging (Orfanos et al., Drug53:358-388, 1997). Retinol is also used in cosmetic formulations toreduce wrinkles and treat cellulite (Orfanos et al., Drug 53:358-388,1997). However, the use of retinol for cosmetic and dermatologicalapplications is severely limited by its poor physicochemical propertiesand skin irritation potential (Melo et al., J Control Release 138:32-39,2009; Kim et al., Toxicol Lett 146:65-73, 2003).

Retinol is lipophilic molecule (Log P 6.20), with poor water solubilityand limited skin permeability. Furthermore, it is highly unstable inpresence of light and moisture (see U.S. Pat. No. 5,851,538 (Froix etal.)) herein incorporated by reference in its entirety. The topicalapplication of retinol causes severe local irritation manifested as milderythema and stratum corneum peeling, leading to non-compliance amongusers (Kim et al., Toxicol Lett 146:65-73, 2003). Applicants havesuccessfully addressed the delivery issues of retinol by encapsulatingretinol in novel protein based micelles for topical application.

Novel nanocarriers have been developed from the corn protein zein, asdescribed herein. One nanocarrier includes conjugating polyethyleneglycol (PEG) to zein. The PEGylated zein forms self-assemblednanomicelles with a hydrophobic core and a hydrophilic shell. Zeindisplays hydrophobicity to skin keratin (Deo et al., Langmuir19:5083-5088, 2003) and hence is a promising carrier for skinapplications. Because zein is hydrophobic, it can be used to encapsulatehydrophobic retinoids inside nanoparticles (see, for example, WO2009/137112, which is incorporated herein by reference in its entirety),or the micelles described herein can be used to encapsulate hydrophobicretinoids to provide a water removable formulation of the retinoid.

Applicants have prepared retinol loaded nanomicelles are in the sizerange of 180-220 nm with an encapsulation efficiency of 79-91%.Encapsulation of retinol in the micelles resulted in a water solubleformulation.

PEG-zein nanomicelles significantly enhanced the solid state and liquidstate stability of retinol against moisture and light induceddegradation. Retinol release was sustained up to 2 days from thePEG-zein nanomicelles.

PEG-zein nanomicelles enhanced the skin penetration of retinol comparedto free retinol aqueous dispersion. Further, PEG-zein nanomicelles canbe used to retain retinol in the skin layers for cosmetic anddermatological applications. A unique aspect of nanocarriers is theability of the nanomicelles to address multiple market challenges fortopical delivery of retinol. These challenges include providing 1) watersoluble and water dispersible formulations of retinol, 2) enhancedstability of retinol against light and moisture induced degradation, 3)a freely flowing, colorless and non-hygroscopic powder of retinol, 4)sustained release formulations of retinol, 5) higher skin penetrationand higher skin retention of retinol, and 6) non-irritating formulationsof retinol.

Zein is a biodegradable US-FDA approved protein polymer with similarcharacteristics to skin keratin and is therefore a skin compatiblenanocarrier. PEG is a US-FDA approved water soluble polymer. PEG-zeinnanomicelles therefore provide a water washable topical formulation forretinol. The amphiphilic PEG-zein micelles serve as a carrier fortransporting hydrophobic drugs such as retinol through the alternatehydrophobic and hydrophilic environment in the skin.

Retinol water solubility is significantly increased after encapsulationin nanomicelles. The retinol release can be sustained from zeinnanomicelles leading to lower dose and reduced frequency of application.The encapsulation of retinol in zein nanomicelles significantlyincreases the shelf-life of retinol formulations. PEG-zein nanomicellesincrease the flowability and dispersibility of retinol in solid andsemi-solid formulations. Because retinol is a hygroscopic sticky powder,the encapsulation of retinol in nanomicelles can overcome the difficulthandling and processing issues associated with retinol.

PEG-zein nanomicelles can enhance the skin penetration and retention ofretinol in the layers of the skin for cosmetic and dermatologicalapplications. Zein nanomicelles can enhance the skin penetration andretention of retinol in layers of the skin for cosmetic anddermatological applications. Encapsulation of retinol in nanomicellesmasks the yellow color of retinol. This improves the aesthetic appeal ofretinol formulations and prevents yellow staining. The lyophilizedPEG-zein nanomicelles can be easily incorporated various topicalformulation matrices, such as gels, creams, lotions and ointments.

The skin penetration studies were carried out with excised pig skin,which is similar to human skin in many important respects (Simon andMaibach, Skin Pharmacol Appl Physiol 13:229-234, 2000). In vivo studiesin mice further demonstrate the ability of the nanomicelles to overcomethe skin irritation of retinol. Advantages of using the nanomicelles inplace of current commercial formulations include:

1. Solubilization.

Retinol is a water insoluble hydrophobic compound. The encapsulation ofretinol in PEG-zein nanomicelles is a water soluble/dispersible. Hencenanomicelles can be used to develop water washable retinol formulationfor topical applications. Generally water washable formulation ispreferred for cosmetic and dermatological applications.

2. Stabilization.

Retinol is highly unstable in presence of moisture and light. Thislimits the shelf-life of retinol formulations and efficacy of theformulation during application. Encapsulation of retinol in PEG-zeinnanomicelles can significantly enhance the stability and shelf-life ofretinol formulations.

3. Sustained Release.

Retinol release can be sustained from PEG-zein nanomicelles. Release canbe sustained from 2 days to a week. This reduces the dose and frequencyof application of retinol.

4. Skin Penetration and Retention.

Retinol has poor skin penetration properties. Nanomicelles lead toenhanced skin penetration of retinol. Depending on the application,retinol can be retained in layers of the skin using nanomicelles forvarious dermatological/cosmetic applications.

5. Cosmecutical Applications.

Retinol loaded micelles can be used for cosmetic applications such asanti-aging, anti-wrinkle, and cellulite treatments.

6. Dermatological Applications.

Retinol loaded nanomicelles can be used for various dermatologicalconditions such as psoriasis, acne, wound-healing and cutaneousmalignancies, such as skin cancer and melanoma.

7. Efficacious and Safe Formulation.

Use of retinol loaded nanomicelles results in more efficacioustreatments. Furthermore, the encapsulation of retinol in thenanomicelles significantly reduces the skin irritation caused byretinol. Skin irritation of retinol is a major issue for non-compliancefor cosmetic and dermatological applications of retinol.

8. Platform Technology for Encapsulation of Other Retinoids.

Various retinoids including retinol, retinoic acid, and theirderivatives, can be encapsulated in prolamine nanomicelles for cosmeticand dermatological applications. Examples of various retinoids suitablefor encapsulation include, but are not limited to, retinol, retinoicacid (such as 13-trans-retinoic acid (tretinoin), 13-cis-retinoic acid(isotretinoin), 9-cis-retinoic acid (alitretinoin)), retinaldehyde,etretnate, acitretin, retinol palmitate, and carotenoids such as acarotene, β-carotene, γ-carotene, β-cryptozanthin, lutein, andzeaxanthin.

9. Combination Therapies.

Retinol nanomicelles can be incorporated into other products, such assunscreens, anti-psoriatic, anti-acne and skin-cancer products alongwith other drugs. Since retinol is encapsulated it will prevent theinteraction with other agents. Other agents such as anti-oxidants,free-radical scavengers, anti-inflammatory agents can also beencapsulated along with retinol in nanomicelles.

Retinol Loaded PEG-Zein Nanomicelles.

Retinol (C201-1300; 286.45 g/mol) has a melting point of 61-63° C., anactivity of 3100 units/mg, and a Log P of 6.2. Retinol is practicallyinsoluble in water, is soluble or partly soluble in ethanol, and ismiscible with chloroform, ether and petroleum spirits.

Retinol is a cosmecutical/therapeutic agent used for various skinconditions including photoaging, acne, wound healing, melasma psoriasis,skin cancer, melanoma and other skin conditions (Orfanos et al., Drug53:358-388, 1997). Retinol has poor water solubility and poorphotostability (Melo et al., J Control Release 138:32-39, 2009; U.S.Pat. No. 5,851,538 (Froix et al.) herein incorporated by reference inits entirety). In addition, it also causes skin irritation (Kim et al.,Toxicol Lett 146:65-73, 2003). Applicants have developed new zein basednanoparticulate topical formulations of retinol. Because zein hassimilar characteristics to skin keratin, it is used as a model proteinto test the skin irritation of excipients used in topical formulations(zein test). Due to its similarity to skin keratin, zein nanocarriersare excellent delivery vehicles for retinol. In addition, PEG is awidely used material in skin formulations. Therefore, the combination ofhydrophobic zein and hydrophilic PEG in PEG-zein micelles is anamphiphilic carrier that enables the transport of molecules through theskin via the alternate hydrophobic and hydrophilic regions in the skin.

This example demonstrates the preparation and characterization ofretinol loaded zein nanomicelles, the improved solubility of retinolusing PEG-zein nanomicelles, the improved stability of retinol byencapsulating in PEG-zein nanomicelles, the sustained release of retinolfrom zein micelles, the ability of zein nanomicelles to enhance skinpenetration and skin retention of retinol, and the lack of or reducedskin irritation of the retinol micelle formulations compared to retinolitself

1. Preparation of the Retinol Loaded Nanomicelles.

PEG-zein, retinol and BHT were dissolved in 90% ethanol and incubatedovernight at 37° C. Later the dispersion was dialyzed against deionizedwater to remove the free retinol. Retinol loaded micelles were thenlyophilized. Radiolabeled (³H) retinol along with ‘cold’ retinol wasused in this study. The size of the nanomicelles was about 180-220 nmand the encapsulation efficiency was 79 to 91%, depending on thePEG-zein/drug ratio and BHT concentration. In absence of BHT, theencapsulation efficiency was <35%. Table 6(10)-1 provides data for thecharacterization of retinol-loaded PEGylated zein micelles preparedusing a dialysis method (e.g., see FIG. 38) and a thin film method(e.g., see FIG. 39), respectively.

TABLE 10-1 Characteristics of retinol-loaded PEGylated zein micellesprepared using a dialysis method and a thin film method, respectively.Retinol BHT Sample (% (% Particle size Encapsulation No. w/w) w/w) (nm)PDI Efficiency (%) Dialysis method 1 0.05 . . . 225.3 ± 8.3 0.374 ± 0.0528.7 ± 3.8 2 0.01 . . . 194.4 ± 5.4 0.390 ± 0.06 28.2 ± 2.2 3 0.015 . .. 231.5 ± 9.9 0.459 ± 0.05 33.1 ± 2.8 4 0.02 . . . 232.2 ± 9.7 0.813 ±0.10 35.0 ± 2.2 5 0.005 0.005 192.2 ± 7.5 0.269 ± 0.03 90.8 ± 3.5 6 0.010.01 191.3 ± 5.9 0.272 ± 0.06 83.3 ± 3.1 7 0.015 0.015 186.0 ± 7.7 0.285± 0.03 80.8 ± 2.5 8 0.02 0.02 197.0 ± 6.7 0.206 ± 0.02 78.6 ± 1.9 9 0.020.04  189.5 ± 10.3 0.322 ± 0.07  77.1 ± 6.44 Film method 1 0.015 0.015 791.5 ± 67.1 0.714 ± 0.1   75.4 ± 8.67 Results are representative oftriplicate samples (average ± SD); PDI = polydispersity index.

2. Increased Solubility/Dispersibility of Retinol in Aqueous Solution.

Free retinol was not dispersible in water and settled at the bottom ofthe vial after attempted dispersion of the agent (FIG. 40). On the otherhand, retinol loaded PEG-zein nanomicelles easily dispersed in water.The solubility of retinol in phosphate buffer (pH 7.4) was significantlyenhanced after encapsulation in nanomicelles. A 10 μg/mL sample ofretinol (retinol equivalent) retinol micelles in phosphate buffer (pH7.4) showed comparable UV absorbance (320 nm) to 10 μg/mL of freeretinol in 20% methanol. Very little absorbance was observed in the 10μg/mL dispersion of retinol in phosphate buffer (pH 7.4).

3. Release of Retinol from Peg-Zein Nanomicelles.

Release studies of the retinol from nanomicelles were carried out inphosphate buffer saline (PBS; pH 7.4). The concentration of retinol wasanalyzed using UV Spectrophotometer at 320 nm, and the release studieswere carried out in triplicate. Retinol release was sustained for up to48 hours from the nanomicelles as shown in FIG. 41.

4. Stability of Retinol Loaded Zein Nanomicelles.

Retinol is a yellow colored powder. It is hygroscopic at ambientconditions and quickly becomes sticky. The encapsulated retinol iscolorless and free flowing, and is far less hygroscopic (FIG. 42). Theretinol sample shown in FIG. 42 was bright yellow and the nanomicelleformulation was white, demonstrating that encapsulation masks the brightyellow color of retinol. The nanomicelle formulation also resulted in amore free flowing powder than pure retinol.

The stability of retinol nanomicellar formulations under ambientconditions and in dark was studied for a period of one week. The solidstability of retinol and retinol loaded nanomicelles (lyophilizedpowder) were also studied for one week. For liquid state stability, freeretinol or retinol loaded nanomicelles was dispersed in phosphate buffer(pH 7.4) and the retinol concentration was measured for a week using aUV spectroscopy method (at 320 nm). Retinol was found to follow firstorder kinetics and the half-life was determined The following resultswere obtained as shown in Tables 10-3 and 10-4 and FIGS. 43, 44, 45, and46.

PEG-zein nanomicelles protected retinol against photodegradation andmoisture induced degradation. The encapsulated retinol showed enhancedstability compared to free retinol in the solid state and in liquidstate. Inclusion of BHT as an antioxidant further enhanced the stabilityof encapsulated retinol. Finally, the shelf-life of retinol wassignificantly enhanced by encapsulation in nanomicelles.

TABLE 10-3 Solid state stability of free and encapsulated retinol.Substance Light (t_(1/2) in hrs) Dark (t_(1/2) in hrs) Retinol solid52.75 63 Retinol micelles 86.63 112 Retinol micelles with BHT 173.251155

TABLE 10-4 Liquid state stability of free and encapsulated retinol inphosphate buffer (pH 7.4). Substance Light (t_(1/2) in hrs) Dark(t_(1/2) in hrs) Retinol 16.11 20.83 Retinol + BHT 35.25 43.42 Retinolmicelles 37 94.88 Retinol micelles with BHT 99 219.5

5. Skin Penetration of Retinol and Encapsulated Retinol.

The skin penetration of retinol and encapsulated retinol was studiedusing excised porcine ear skin using a Franz diffusion cell.Radiolabeled (³H) retinol along with ‘cold’ retinol was used in thisstudy. The amount of retinol in the skin homogenate and receptor mediumat the end of 48 hours was estimated using radiochemical analysis. Theexperiments were repeated 6 times (±SD). As can been seen in FIG. 47,the encapsulated retinol resulted in greater retention of retinol in theskin. Micelles resulted in approximately 5 fold increased skin retentionof retinol. The ratio of “retinol in skin to receptor” was 3 and 6.5,for free retinol and retinol micelles, respectively. The results showthat micelles increased the overall skin penetration and retention ofretinol.

To demonstrate the follicular targeting of retinol a skin sandwich modelwas used. In the sandwich skin (see FIG. 48), the follicular pathwaysare blocked by the stratum corneum sandwiched over the epidermis. In thesandwich skin model the amount of retinol transported into the receptorcompartment was reduced both for free and micelle encapsulated retinolcompared to conventional skin epidermis penetration studies. However,there was significant reduction in the transport of retinol from themicelles indicating that a significant fraction of retinol micelles istransported through the hair follicles. Given the use of retinol totreat acne (acne mainly originates from the hair follicles), the retinolmicelles will have the advantage of targeting retinol to the diseasesite in the hair follicles.

In summary, PEG-zein nanomicelles significantly increased the aqueoussolubility and dispersibility of retinol. Encapsulation of retinol innanomicelles resulted in a free flowing colorless powder, unlike freeretinol, which is a yellow, sticky and hygroscopic powder. Zeinnanomicelles effectively sustained the release of retinol.Photostability and hydrolytic stability of retinol is significantlyenhanced by encapsulating in zein nanomicelles, which was furtherenhanced by addition of BHT as an antioxidant, and PEG-zein nanomicellesresulted in higher skin retention of retinol. The nanomicelles can alsoreduce the skin irritation of retinol.

Preparation of a Cream Formulation for Retinol Micelles.

To demonstrate the feasibility of a skin formulation for delivery forcommercial development, a commercial cream base (MEDCO Labs) was used toincorporate free retinol or retinol encapsulated in zein nanomicelles.Cream base contains stearyl alcohol (14%, cetyl esters was (3.5%),glyceryl monostearate (2%), polyoxyethylene stearyl ether (3%), sorbitol(10%), isopropyl palmitate (2%), methyl paraben (0.16%), propyl paraben(0.4%) and purified water (65%). Retinol equivalent to 0.1% was weighedand transferred to watch glass and mixed homogenously using glass rod bygeometric dilution. Other formulation, including but not limited to,oil-water cream, water in oil cream, ointment, gel, and the like may beused. The mixture was spiked with 0.05 μCi of ³H-retinol and mixedthoroughly in the cream. Finally, the prepared cream formulations weretransferred to glass vials and stored until use.

TABLE 11 Retinol cream formulations Retinol (0.1% w/w) cream - 1 gRetinol  0.001 g Cream base  0.800 g Retinol micelle cream (0.1% w/w)cream - 1 g Retinol micelles 0.0625 g Cream base 0.9375 g

Stability of retinol micellar cream formulation was measured for aperiod of one-month (see FIG. 49). As shown in the Figure, theformulation remained stable and did not show any degradation at roomtemperature.

In Vitro Release of Retinol from Cream Formulations.

About 40 mg of the cream base and micelle cream were place in a verticaldiffusion cell dialysis membrane (MWCO ˜8,000-10,000 Da) for the releasestudy, the receptor medium consisted of pH 7.4 buffer. Samples werecollected from the receptor medium and analyzed by radiochemical methodusing ³H retinol. Each data point represents mean±SD (n=3). As can beseen in FIG. 50, more retinol is released from the plain cream compareto the micelle cream.

In Vitro Skin Penetration.

Excised human skin was sandwiched between the two compartments of avertical diffusion cell. The receptor medium consisted of phosphatebuffer (pH 7.4) maintained at 37° C. and stirred using a magnetic bead.Free or retinol encapsulated micelle cream formulation was loaded in thedonor chamber. The formulation was applied for 6 hours and then theformulation was removed and the penetration study was continued for 48hours. At the end of the study, the retinol concentration in the skinand receptor compartment was measured by radiochemical method using ³Hlabeled retinol. The skin was digested using 0.1M sodium hydroxide todetermine the retinol concentration. As can be seen in FIG. 51, for theplain cream, more retinol was present in the receptor compartment thanin the skin. In contrast, the micelle cream showed the opposite, wheremore retinol was found in the skin than in the receptor compartment.

Skin Irritation of Retinol and Encapsulated Retinol Cream Formulation.

The skin irritation of standard vs. encapsulated formulations can betested in vivo in SKH-1 hairless mice using treatments groups as listedin Table 12.

TABLE 12 Treatment groups for a skin irritation study. Groups TreatmentGroup 1 Control (no treatment) Group 2 Retinol cream Group 3 BlankPEG-zein micelles cream Group 4 Retinol nanomicelles cream Group 5Sodium lauryl sulfate cream (positive control)

The retinol cream formulations (0.5 g of 0.1% w/v retinol equivalent)were applied to the backs of SKH-1 hairless mice everyday for five (5)days. The transepidermal water loss (TEWL) values were measured using anTEWA meter (Delfin) every day before applying the formulation. Theincrease in TEWL is a measure of skin irritation and as can be seen inFIG. 52, the retinol encapsulated in micelles showed no skin irritationand was comparable to negative control (i.e., no treatment). On theother hand the free retinol cream shows skin irritation. Sodium laurylsulfate (SLS), a known skin irritant, was used as the positive control.

In Vivo Topical Bioavailability.

The cream formulations were applied on the back skin of mice underisoflurane anesthesia. After euthanizing the animals, the skin wastape-stripped using SCOTCH TAPE to remove stratum corneum. The amount ofretinol in skin (stratum corneum and epidermis/dermis) and blood weredetermined using ³H retinol by radiochemical analysis. As can be seen inFIG. 53, the micelle encapsulated retinol was retained in the skin withno systemic absorption into the blood. Values are mean±SD (n=3).

Example 7 Casein Micelles

Casein is a milk protein that can form micelles under appropriateconditions. Although some studies have described the use of caseinmicelles as a delivery vehicle, casein micelles have not been used asdelivery agents for skin applications. Casein can be combined withPEG-zein to form novel mixed micelles.

The general steps for preparing retinol loaded 3-casein micelles are asfollows. β Casein (20 mg) and retinol (0.1 mg in 600 μL of ethanol) maybe dissolved in 10 mL of 0.1M PBS pH 7.0. The mixture may be incubatedovernight at about 37° C., followed by lyophilization (e.g., for about24 hours) at ˜100° C. under 100 mTorr vacuum. The resulting micellepowder may be stored in a dessicator at about 2-8° C. for an extendedperiod of time. Table 13 below illustrates various characteristics ofretinol-loaded β-casein micelles. For the preparation of retinol loadedβ-casein micelle, retinol concentrations can range from about 0.005 toabout 0.05% w/w. 3-Casein concentrations ranged from about 0.15-0.25%w/v.

TABLE 13 Characteristics of retinol-loaded β-casein micelles. SampleRetinol Particle Encapsulation No. (% w/w) BHT (% w/w) size (nm) PDIEfficiency (%) 1 0.005 — 207.4 0.656 9.74 2 0.015 — 109.1 0.616 10.28 30.005 0.005 76.9 0.626 10.25 4 0.015 0.015 68.9 0.510 11.06

Casein can also be used to prepare Nile red containing micelles (see,FIG. 54). Table 14 provides characteristics of such micelles.

TABLE 14 Particle Size Encapsulation Sample name (nm) PI Efficiency (%)Nile red-casein micelles 245 ± 15 nm 0.38 ± 0.41 78 ± 5%

As seen in FIG. 55, the encapsulation of Nile red in the casein micellessignificantly increased the skin penetration of Nile red. Excisedporcine skin was sandwiched between the two compartments of a verticaldiffusion cell. The receptor medium consisting of phosphate buffer (pH7.4) was maintained at 37° C. and stirred using a magnetic bead. Free orencapsulated Nile red was applied on the skin for 6 hours. At the end ofthe study, the skin was washed and observed under a confocalfluorescence microscope. The fluorescence in the SC (0-15 μm) and viableepidermis (20-100 μm) was quantified using IMAGEJ software. Each valueis avg.±SD (n=4). Significant difference at p<0.05.

Example 8 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or cosmeticadministration of a micelle formulation described herein, which can bean aqueous dispersion or a lyophilized powder (hereinafter referred toas ‘Composition X’):

(i) Tablet 1 mg/tablet ‘Composition X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0 (ii) Tablet 2 mg/tablet ‘Composition X’ 20.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg/capsule ‘CompositionX’ 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinizedstarch 120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg/mL)mg/mL ‘Composition X’ 1.0 Dibasic sodium phosphate 12.0 Monobasic sodiumphosphate 0.7 Sodium chloride 4.5 1.0N Sodium hydroxide solution q.s.(pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (v)Injection 2 (10 mg/mL) mg/mL ‘Composition X’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.00.1N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL (vi) Aerosol mg/can ‘Composition X’ 20 Oleic acid10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000 (vii) Topical Gel 1 wt. % ‘CompositionX’ 5% Carbomer 934 1.25%   Triethanolamine q.s. (pH adjustment to 5-7)Methyl paraben 0.2%   Purified water q.s. to 100 g (viii) Topical Gel 2wt. % ‘Composition X’ 5% Methylcellulose 2% Methyl paraben 0.2%   Propylparaben 0.02%   Purified water q.s. to 100 g (ix) Topical Ointment wt %‘Composition X’ 5% Propylene glycol 1% Anhydrous ointment base 40% Polysorbate 80 2% Methyl paraben 0.2%   Purified water q.s. to 100 g (x)Topical Cream 1 wt. % ‘Composition X’ 5% White bees wax 10%  Liquidparaffin 30%  Benzyl alcohol 5% Purified water q.s. to 100 g (xi)Topical Cream 2 wt. % ‘Composition X’ 5% Stearic acid 10%  Glycerylmonostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropylpalmitate 2% Methyl Paraban 0.2%   Purified water q.s. to 100 g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Composition X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. Although the invention hasbeen described with reference to the above examples, it will beunderstood that modifications and variations are encompassed within thespirit and scope of the invention. Accordingly, the invention is limitedonly by the following claims.

1. A stable micelle comprising an amphiphilic copolymer containing atleast one hydrophobic moiety covalently conjugated to at least onehydrophilic moiety, wherein said at least one hydrophobic moiety is aprolamine protein selected from the group consisting of zein, gliadin,hordein, kafirin, and combinations thereof, wherein the critical micelleconcentration (CMC) of the copolymer in water is between about 0.015 g/Lto about 0.035 g/L, and wherein said stable micelle has a biodegradablehydrophilic shell-hydrophobic core structure.
 2. The stable micelle ofclaim 1, wherein the stable micelle is formed from hydration of a filmcomprising said amphiphilic copolymer or the stable micelle is formedfrom a dialysate comprising said amphiphilic copolymer, and wherein saidamphiphilic copolymer is a block copolymer or a graft copolymer.
 3. Thestable micelle of claim 1, wherein the at least one hydrophobic moietyis a zein protein and the at least one hydrophilic moiety is a PEG, andwherein at least about 50% of surface amino groups of the zein proteinare PEGylated.
 4. The stable micelle of claim 3, wherein the PEG has amolecular weight of at least 3,000 Da.
 5. The stable micelle of claim 4,wherein the polydispersity index (PDI) of the stable micelle is lessthan about 0.5 when the molecular weight of PEG is greater than about2000 Da.
 6. The stable micelle of claim 3, wherein the molecular weightof PEG is between about 1 kDa and 220 kDa.
 7. The stable micelle ofclaim 3, wherein the micelle particle size is from about 10 nm to about300 nm.
 8. The stable micelle of claim 1, further comprising one or morecargo molecules, wherein said one or more cargo molecules areencapsulated within the hydrophobic core, covalently or non-covalentlycomplexed to the hydrophobic moiety, covalently or non-covalentlycomplexed to the hydrophilic moiety, or a combination thereof.
 9. Thestable micelle of claim 8, wherein the one or more cargo molecules areselected from the group consisting of a drug, a protein, a nucleic acid,a hormone, a receptor, a diagnostic agent, an imaging agent, and acombination thereof.
 10. The stable micelle of claim 9, wherein the drugis a hydrophobic drug having a Log P of about 1 to
 7. 11. The stablemicelle of claim 9, wherein the drug is an anti-oxidant,anti-inflammatory, or an anticancer drug, and wherein the drug deliveredvia the stable micelle exhibits lower toxicity and enhanced efficacycompared to the drug delivered in the absence of said stable micelle.12. The stable micelle of claim 11, wherein the drug is curcumin ordoxorubicin.
 13. The stable micelle of claim 9, wherein the imagingagent is Nile red.
 14. The stable micelle of claim 9, wherein the drugis a retinoid.
 15. The stable micelle of claim 14, wherein the retinoidis selected from the group consisting of retinol, 13-cis-retinoic acid,13-trans-retinoic acid, retinaldehyde, tretinoin, isotretinoin,etretnate, acitretin, retinyl palmitate, α-carotene, β-carotene,γ-carotene, β-cryptozanthin, lutein, zeaxanthin, and a combinationthereof, and wherein the photostability and hydrolytic stability of theretinoid in the hydrophobic core is enhanced compared to free retinoid.16. The stable micelle of claim 14, further comprising butylatedhydroxyltoluene (BHT), casein, or a combination thereof.
 17. A method ofpreparing a stable micelle comprising: dissolving a prolamine proteinand a monoalkylated polyethylene glycol (mPEG) in a hydroalcoholicsolvent to form a first mixture; heating the first mixture to formcovalently conjugated PEGylated prolamine and optionally quenchingexcess reactive groups of the PEG in the hydroalcoholic suspension;adding a buffer to the first mixture to precipitate the PEGylatedprolamine from the hydroalcoholic solvent and dialyzing the PEGylatedprolamine against an aqueous solution; lyophilizing the resultingdialysate; dissolving the lyophilized PEGylated-prolamine in ahydroalcoholic solvent to form a second mixture; and either: (a)dialyzing the second mixture against an aqueous buffer to form a stablemicelle or (b) evaporating the second mixture to form a dry film,hydrating the film with an aqueous buffer, and sonicating the hydratedfilm to form a stable micelle, wherein the critical micelleconcentration (CMC) of the PEGylated prolamine in water is between about0.015 g/L to about 0.035 g/L.
 18. The method of claim 17, furthercomprising lyophilizing the stable micelle to form a dry powder.
 19. Themethod of claim 17, further comprising adding one or more cargomolecules dissolved in a solvent system to the first mixture, resultingin the formation of a plurality of stable micelles loaded with one ormore cargo molecules, wherein said one or more cargo molecules areencapsulated within the hydrophobic core, covalently or non-covalentlycomplexed to the hydrophobic moiety, covalently or non-covalentlycomplexed to the hydrophilic moiety, or a combination thereof, andwherein said plurality of stable micelles enhance a property of the oneor more cargo molecules contained therein, which property is selectedfrom the group consisting of enhancing the water solubility one or morecargo molecules, enhancing the chemical stability of one or more cargomolecules, enhancing the drug efficacy of one or more cargo molecules,enhancing the shelf life of one or more cargo molecules, enhancing thetumor accumulation of one or more cargo molecules, enhancing the skinpenetration of one or more cargo molecules, and combinations thereof,compared to the property of said one or more cargo molecules in theabsence of said plurality of stable micelles.
 20. A method of treating aP-glycoprotein (P-gp)-dependent multidrug resistant (MDR) type cancer, askin or follicular disorder in subject in need thereof comprisingadministering the stable micelle of claim 1.